Amyloid imaging as a surrogate marker for efficacy of anti-amyloid therapies

ABSTRACT

The present method for determining the efficacy of therapy in the treatment of amyloidosis involves administering to a patient in need thereof a compound of formula (I) or formula (II) or structures 1-45 and imaging the patient. After said imaging, at least one anti-amyloid agent is administered to said patient. Then, an effective amount of a compound of formula (I) or formula (II) or structures 1-45 is administered to the patient and the patient is imaged again. Finally, baseline levels of amyloid deposition in the patient before treatment with the anti-amyloid agent are compared with levels of amyloid deposition in the patient following treatment with the anti-amyloid agent.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Divisional Application of U.S. Application Ser.No. 11/666,083 filed Nov. 28, 2007, which is the U.S. National Phase ofPCT/US2005/023617 dated Jul. 1, 2005, which claims priority from U.S.Provisional Application No. 60/584,487 filed Jul. 2, 2004. The subjectmatter of each of the above-referenced applications is incorporatedherein in entirety by reference.

BACKGROUND

Amyloidosis is a diverse group of disease processes characterized byextracellular tissue deposits, in one or many organs, of proteinmaterials which are generically termed amyloid. Amyloid may bedistinguished grossly by a starch-like staining reaction with iodine(thus the term amyloid), microscopically by its extracellulardistribution and tinctorial and optical properties when stained withCongo red, and by its protein fibril structure as shown by electronmicroscopy and x-ray crystallography (see Table-1). Exemplaryamyloidosis diseases are Alzheimer's Disease (“AD”), Down's Syndrome,Type 2 diabetes mellitus, and mild cognitive impairment (MCI).

AD is a neurodegenerative illness characterized by memory loss and othercognitive deficits. McKhann et al., Neurology 34: 939 (1984). It is themost common cause of dementia in the United States. AD can strikepersons as young as 40-50 years of age, yet, because the presence of thedisease is difficult to determine without dangerous brain biopsy, thetime of onset is unknown. The prevalence of AD increases with age, withestimates of the affected population reaching as high as 40-50% by ages85-90. Evans et al., JAMA 262: 2551 (1989), Katzman, Neurology 43: 13(1993).

Neuropathologically, AD is characterized by the presence of neuriticplaques (NP), neurofibrillary tangles (NFT), and neuronal loss, alongwith a variety of other findings. Mann, Mech. Ageing Dev. 31: 213(1985). Post-mortem slices of brain tissue of victims of AD exhibit thepresence of amyloid in the form of proteinaceous extracellular cores ofthe neuritic plaques that are characteristic of AD. The amyloid cores ofthese neuritic plaques are composed of a protein called the β-amyloid(Aβ) that is arranged in a predominately beta-pleated sheetconfiguration.

AD is believed to afflict some 4 million Americans and perhaps 20-30million people worldwide. AD is recognized as a major public healthproblem in developed nations. Several therapeutic targets have emergedfrom the ongoing elucidation of the molecular basis of AD. For example,four cholinesterase inhibitors have been approved for the symptomatictreatment of patients with AD—tacrine (Cognex, Warner-Lambert, MorrisPlains, N.J.); donepezil (Aricept, Eisai, Inc., Teaneck, N.J., andPfizer, Inc., New York, N.Y.); rivagstigmine (Exelon, Novartis, Basel,Switzerland); and galantamine (Reminyl, Janssen, Titusville, N.J.).Potential new AD therapies that are currently being developed involveimmunotherapy, secretase inhibitors or anti-inflammatory drugs. However,to date, there are no available drugs proven to modify the course ofcognitive decline.

A major hurdle to developing anti-amyloid therapies is exemplified bythe following quote from (Hock, C. et al., 2003, Neuron, 38:547-554),directed to use of immunotherapy as an anti-amyloid therapy: “[w]e donot know whether brain Aβ-amyloid load was reduced in our studypatients; in vivo imaging techniques will be required to answer thisquestion.” The ability to quantify amyloid load before treatment andthen follow the effects of treatment is critical to the efficientdevelopment of this class of drugs. The present invention employsamyloid imaging as a surrogate marker of efficacy for anti-amyloidtherapies.

SUMMARY OF THE INVENTION

The present invention is directed to a method of determining theefficacy of therapy in the treatment of amyloidosis, comprising:

-   (A) administering to a patient in need thereof an effective amount    of a compound of the following formula:

wherein

(i) Z is S, NR′, O or C(R′)₂, such that when Z is C(R′)₂, the tautomericform of the heterocyclic ring may form an indole:

wherein R′ is H or a lower alkyl group,

(ii) Y is NR¹R², OR², or SR²,

(iii) R¹ is selected from the group consisting of H, a lower alkylgroup, (CH₂)_(n)OR′ (wherein n=1, 2, or 3), CF₃, CH₂—CH₂X, CH₂—CH₂—CH₂X(wherein X=F, Cl, Br or I), (C═O)—R′, R_(ph), and (CH₂)_(n)R_(ph)(wherein n=1, 2, 3, or 4 and R_(ph) represents an unsubstituted orsubstituted phenyl group with the phenyl substituents being chosen fromany of the non-phenyl substituents defined below for R³-R¹⁰ and R′ is Hor a lower alkyl group);

(iv) R² is selected from the group consisting of H, a lower alkyl group,(CH₂)_(n)OR′ (wherein n=1, 2, or 3), CF₃, CH₂—CH₂X, CH₂—CH₂—CH₂X(wherein X=F, Cl, Br or I), (C═O)—R′, R_(ph), and (CH₂)_(n)R_(ph)(wherein n=1, 2, 3, or 4 and R_(ph) represents an unsubstituted orsubstituted phenyl group with the phenyl substituents being chosen fromany of the non-phenyl substituents defined below for R³-R¹⁰ and R′ is Hor a lower alkyl group);

(v) R³ is selected from the group consisting of H, F, Cl, Br, I, a loweralkyl group, (CH₂)_(n)OR′ (wherein n=1, 2, or 3), CF₃, CH₂—CH₂X,O—CH₂—CH₂X, CH₂—CH₂—CH₂X, O—CH₂—CH₂—CH₂X (wherein X=F, Cl, Br or I), CN,(C═O)—R′, N(R′)₂, NO₂, (C═O)N(R′)₂, O(CO)R′, OR′, SR′, COOR′, R_(ph),CR′═CR′—R_(ph), CR₂′—CR₂′—R_(ph) (wherein R_(ph) represents anunsubstituted or substituted phenyl group with the phenyl substituentsbeing chosen from any of the non-phenyl substituents defined for R¹-R¹⁰and wherein R′ is H or a lower alkyl group) and a tri-alkyl tin;

(vi) R⁴ is selected from the group consisting of H, F, Cl, Br, I, alower alkyl group, (CH₂)_(n)OR′ (wherein n=1, 2, or 3), CF₃, CH₂—CH₂X,O—CH₂—CH₂X, CH₂—CH₂—CH₂X, O—CH₂—CH₂—CH₂X (wherein X=F, Cl, Br or I), CN,(C═O)—R′, N(R′)₂, NO₂, (C═O)N(R′)₂, O(CO)R′, OR′, SR′, COOR′, R_(ph),CR′═CR′—R_(ph), CR₂′—CR₂′—R_(ph) (wherein R_(ph) represents anunsubstituted or substituted phenyl group with the phenyl substituentsbeing chosen from any of the non-phenyl substituents defined for R¹-R¹⁰and wherein R′ is H or a lower alkyl group) and a tri-alkyl tin;

(vii) R⁵ is selected from the group consisting of H, F, Cl, Br, I, alower alkyl group, (CH₂)_(n)OR′ (wherein n=1, 2, or 3), CF₃, CH₂—CH₂X,O—CH₂—CH₂X, CH₂—CH₂—CH₂X, O—CH₂—CH₂—CH₂X (wherein X=F, Cl, Br or I), CN,(C═O)—R′, N(R′)₂, NO₂, (C═O)N(R′)₂, O(CO)R′, OR′, SR′, COOR′, R_(ph),CR₂′—CR₂′—R_(ph) (wherein R_(ph) represents an unsubstituted orsubstituted phenyl group with the phenyl substituents being chosen fromany of the non-phenyl substituents defined for R¹-R¹⁰ and wherein R′ isH or a lower alkyl group) and a tri-alkyl tin;

(viii) R⁶ is selected from the group consisting of H, F, Cl, Br, I, alower alkyl group, (CH₂)_(n)OR′ (wherein n=1, 2, or 3), CF₃, CH₂—CH₂X,O—CH₂—CH₂X, CH₂—CH₂—CH₂X, O—CH₂—CH₂—CH₂X (wherein X=F, Cl, Br or I), CN,(C═O)—R′, N(R′)₂, NO₂, (C═O)N(R′)₂, O(CO)R′, OR′, SR′, COOR′, R_(ph),CR′═CR′—R_(ph), CR₂′—CR₂′—R_(ph) (wherein R_(ph) represents anunsubstituted or substituted phenyl group with the phenyl substituentsbeing chosen from any of the non-phenyl substituents defined for R¹-R¹⁰and wherein R′ is H or a lower alkyl group) and a tri-alkyl tin;

(ix) R⁷ is selected from the group consisting of H, F, Cl, Br, I, alower alkyl group, (CH₂)_(n)OR′ (wherein n=1, 2, or 3), CF₃, CH₂—CH₂X,O—CH₂—CH₂X, CH₂—CH₂—CH₂X, O—CH₂—CH₂—CH₂X (wherein X=F, Cl, Br or I), CN,(C═O)—R′, N(R′)₂, NO₂, O(CO)R′, OR′, SR′, COOR′, R_(ph), CR′═CR′—R_(ph),CR₂′—CR₂′—R_(ph) (wherein R_(ph) represents an unsubstituted orsubstituted phenyl group with the phenyl substituents being chosen fromany of the non-phenyl substituents defined for R¹-R¹⁰ and wherein R′ isH or a lower alkyl group) and a tri-alkyl tin;

(x) R⁸ is selected from the group consisting of H, F, Cl, Br, I, a loweralkyl group, (CH₂)_(n)OR′ (wherein n=1, 2, or 3), CF₃, CH₂—CH₂X,O—CH₂—CH₂X, CH₂—CH₂—CH₂X, O—CH₂—CH₂—CH₂X (wherein X=F, Cl, Br or I), CN,(C═O)—R′, N(R′)₂, NO₂, (C═O)N(R′)₂, O(CO)R′, OR′, SR′, COOR′, R_(ph),CR′═CR′—R_(ph), CR₂′—CR₂′—R_(ph) (wherein R_(ph) represents anunsubstituted or substituted phenyl group with the phenyl substituentsbeing chosen from any of the non-phenyl substituents defined for R¹-R¹⁰and wherein R′ is H or a lower alkyl group) and a tri-alkyl tin;

(xi) R⁹ is selected from the group consisting of H, F, Cl, Br, I, alower alkyl group, (CH₂)_(n)OR′ (wherein n=1, 2, or 3), CF₃, CH₂—CH₂X,O—CH₂—CH₂X, CH₂—CH₂—CH₂X, O—CH₂—CH₂—CH₂X (wherein X=F, Cl, Br or I), CN,(C═O)—R′, N(R′)₂, NO₂, (C═O)N(R′)₂, O(CO)R′, OR′, SR′, COOR′, R_(ph),CR′═CR′—R_(ph), CR₂′—CR₂′—R_(ph) (wherein R_(ph) represents anunsubstituted or substituted phenyl group with the phenyl substituentsbeing chosen from any of the non-phenyl substituents defined for R¹-R¹⁰and wherein R′ is H or a lower alkyl group) and a tri-alkyl tin;

(xii) R¹⁰ is selected from the group consisting of H, F, Cl, Br, I, alower alkyl group, (CH₂)_(n)OR′ (wherein n=1, 2, or 3), CF₃, CH₂—CH₂X,O—CH₂—CH₂X, CH₂—CH₂—CH₂X, O—CH₂—CH₂—CH₂X (wherein X=F, Cl, Br or I), CN,N(R′)₂, NO₂, (C═O)N(R′)₂, O(CO)R′, OR′, SR′, COOR′, R_(ph),CR′═CR′—R_(ph), CR₂′—CR₂′—R_(ph) (wherein R_(ph) represents anunsubstituted or substituted phenyl group with the phenyl substituentsbeing chosen from any of the non-phenyl substituents defined for R¹-R¹⁰and wherein R′ is H or a lower alkyl group) and a tri-alkyl tin;

alternatively, one of R³-R¹⁰ may be a chelating group (with or without achelated metal group) of the form W-L or V—W-L, wherein V is selectedfrom the group consisting of —COO—, —CO—, —CH₂O— and —CH₂NH—; W is—(CH₂)_(n) where n=0, 1, 2, 3, 4, or 5; and L is:

wherein M is selected from the group consisting of Tc and Re;

-   (B) imaging said patient; then-   (C) administering to said patient in need thereof at least one    anti-amyloid agent;-   (D) subsequently administering to said patient in need thereof an    effective amount of a compound of formula (I);-   (E) imaging said patient; and-   (F) comparing levels of amyloid deposition in said patient before    treatment with said at least one anti-amyloid agent to levels of    amyloid deposition in said patient after treatment with said at    least one anti-amyloid agent.

In some embodiments, the anti-amyloid agent comprises one or moreantibodies against Aβ peptide.

In other embodiments, the anti-amyloid agent comprises one or moreinhibitors of β- and/or γ-secretase.

In some embodiments, the anti-amyloid agent comprises a small moleculethat binds to Aβ1-42, such as a decoy peptide.

In other embodiments, the amyloidosis is AD.

In some embodiment, the amyloidosis is an amyloid deposition disorder,wherein a preferred embodiment encompasses amyloidosis which is anamyloid plaque deposition disorder.

In some embodiments, the imaging is selected from the group consistingof gamma imaging, magnetic resonance imaging, and magnetic resonancespectroscopy.

In other embodiments, the imaging is done by gamma imaging, and thegamma imaging is PET or SPECT.

In some embodiments, the compound of Formula (I) is:

In other embodiments, the compound of Formula (I) contains a ¹¹C label.

In some embodiments, the anti-amyloid agent is a peripheral sink agent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows binding of 1 nM{N-methyl-³H}2-[4′-(methylamino)phenyl]6-hydroxy-benzothiazole(“[³H]PIB”) to frontal cortex (Fr) and cerebellum (Cb) of control brain(Cntl; n=4; white bars; circles), AD brain (AD; n=5; black bars;squares) and an AN-1792-treated AD case (n=1 repeated×1; hatched bars;triangles). The results indicate that treatment with AN-1792 vaccinedecreases the binding of the amyloid tracer,2-[4′-(methylamino)phenyl]6-hydroxy-benzothiazole (“PIB”), to brainhomogenates.

FIG. 2 shows Aβ42 immunoreactivity (ir) and X-34 histofluorescentlabeling of β-pleated sheet in the temporal cortex of AD patients 572(A,D) and 5180 (B,E), compared to a representative end-stage AD patient(C,F). Scale bar=200 μm. Large areas devoid of plaques in case 572 aremarked with asterisks. Case 5180 is devoid of plaques, but shows someneurofibrollary tangles and neuritic elements stained by X-34.

FIG. 3 shows Aβ42 immunoreactivity and X-34 histofluorescent labeling ofβ-pleated sheet in the frontal cortex of patients 572 (A,D) and 5180(B,E), compared to a representative end-stage Alzheimer's diseasepatient. Scale bar=200 μm. Areas devoid of plaques in case 572 aremarked with asterisks. Case 5180 is devoid of plaques, but shows someneurofibrollary tangles stained by X-34 (see FIG. 4).

FIG. 4 shows X-34 staining of β-pleated sheet-containing neurofibrillarytangles, neuropil threads, dystrophic neurites and senile plaques inpatients 572 and 5180. Note that patient 5180 has abundant neuriticelements, but no plaques. Areas cleared of X-34 stained elements aremarked with asterisks. These cleared areas strongly suggest the presenceof plaques before AN-1792 treatment. Scale bar=100 μm.

FIG. 5: The top graph charts ELISA data for Aβ42 in cases 572 and 5180in frontal, parietal, temporal and cerebellar cortices. These arecompared to published data for the frontal, parietal and temporalcortices of elderly controls and AD subjects (Naslund et al. 2000, Jama283, 1571-1577). The bottom graph charts [³H]PIB binding in cases 572and 5180 in frontal, parietal, temporal and cerebellar cortices,compared to [³H]PIB binding to the same areas of elderly controls (n=4)and AD subjects (n=5). Note that [³H]PIB binding correlates well withELISA and histologic data in FIGS. 2-4.

DETAILED DESCRIPTION

The present invention is directed to a method for determining theefficacy of therapy in the treatment of amyloidosis. The method involvesthe use of amyloid imaging as a surrogate marker. Surrogate markers area special type of biomarker that may be used in place of clinicalmeasurements as a clinical endpoint for drug approval purposes. Thus,the methods described herein are useful in drug development trials. Forexample, the measurement of cholesterol levels is now an acceptedsurrogate marker of atherosclerosis. In addition, the methods areclinically useful in assisting patient management decisions. In thatregard, quantitative evaluations of amyloid burden can improve clinicaldecisions concerning drug dose or treatment selections. The presentinvention involves the use of amyloid imaging as a surrogate marker ofefficacy for anti-amyloid therapies.

The term “amyloidosis” refers to a disease associated with amyloiddeposition, such as Alzheimer's Disease, Down's Syndrome, Type 2diabetes mellitus, hereditary cerebral hemorrhage amyloidosis (Dutch),amyloid A (reactive), secondary amyloidosis, MCI, familial Mediterraneanfever, familial amyloid nephropathy with urticaria and deafness(Muckle-wells Syndrome), amyloid lambda L-chain or amyloid kappa L-chain(idiopathic, myeloma or macroglobulinemia-associated) A beta 2M (chronichemodialysis), ATTR (familial amyloid polyneuropathy (Portuguese,Japanese, Swedish)), familial amyloid cardiomyopathy (Danish), isolatedcardiac amyloid, systemic senile amyloidoses, AIAPP or amylininsulinoma, atrial naturetic factor (isolated atrial amyloid),procalcitonin (medullary carcinoma of the thyroid), gelsolin (familialamyloidosis (Finnish)), cystatin C (hereditary cerebral hemorrhage withamyloidosis (Icelandic)), AApo-A-I (familial amyloidoticpolyneuropathy-Iowa), AApo-A-II (accelerated senescence in mice),fibrinogen-associated amyloid; and Asor or Pr P-27 (scrapie, CreutzfeldJacob disease, Gertsmann-Straussler-Scheinker syndrome, bovinespongiform encephalitis) or in cases of persons who are homozygous forthe apolipoprotein E4 allele, and the condition associated withhomozygosity for the apolipoprotein E4 allele or Huntington's disease.The invention encompasses diseases associated with amyloid plaquedeposition. Preferably, the disease associated with amyloid depositionis AD.

The present method provides a means of evaluating success ofanti-amyloid therapies. In some embodiments, the present method providesa means for evaluating clinical success of anti-amyloid therapies. Insome embodiments, the method may be used to evaluate clinical success inmildly impaired subjects with few or no clinical symptoms to follow. Thebasic method of determining the efficacy of therapy in the treatment ofamyloidosis involves:

(A) administering to a patient in need thereof an effective amount ofcompound of the following formula:

wherein

(i) Z is S, NR′, O or C(R′)₂, such that when Z is C(R′)₂, the tautomericform of the heterocyclic ring may form an indole:

wherein R′ is H or a lower alkyl group,

(ii) Y is NR′R², OR², or SR²,

(iii) R¹ is selected from the group consisting of H, a lower alkylgroup, (CH₂)_(n)OR′ (wherein n=1, 2, or 3), CF₃, CH₂—CH₂X, CH₂—CH₂—CH₂X(wherein X=F, Cl, Br or I), (C═O)—R′, R_(ph), and (CH₂)_(n)R_(ph)(wherein n=1, 2, 3, or 4 and R_(ph) represents an unsubstituted orsubstituted phenyl group with the phenyl substituents being chosen fromany of the non-phenyl substituents defined below for R³-R¹⁰ and R′ is Hor a lower alkyl group);

(iv) R² is selected from the group consisting of H, a lower alkyl group,(CH₂)_(n)OR′ (wherein n=1, 2, or 3), CF₃, CH₂—CH₂X, CH₂—CH₂—CH₂X(wherein X=F, Cl, Br or I), (C═O)—R′, R_(ph), and (CH₂)_(n)R_(ph)(wherein n=1, 2, 3, or 4 and R_(ph) represents an unsubstituted orsubstituted phenyl group with the phenyl substituents being chosen fromany of the non-phenyl substituents defined below for R³-R¹⁰ and R′ is Hor a lower alkyl group);

(v) R³ is selected from the group consisting of H, F, Cl, Br, I, a loweralkyl group, (CH₂)_(n)OR′ (wherein n=1, 2, or 3), CF₃, CH₂—CH₂X,O—CH₂—CH₂X, CH₂—CH₂—CH₂X, O—CH₂—CH₂—CH₂X (wherein X=F, Cl, Br or I), CN,(C═O)—R′, N(R′)₂, NO₂, (C═O)N(R′)₂, O(CO)R′, OR′, SR′, COOR′, R_(ph),CR′═CR′—R_(ph), CR₂′—CR₂′—R_(ph) (wherein R_(ph) represents anunsubstituted or substituted phenyl group with the phenyl substituentsbeing chosen from any of the non-phenyl substituents defined for R¹-R¹⁰and wherein R′ is H or a lower alkyl group) and a tri-alkyl tin;

(vi) R⁴ is selected from the group consisting of H, F, Cl, Br, I, alower alkyl group, (CH₂)_(n)OR′ (wherein n=1, 2, or 3), CF₃, CH₂—CH₂X,O—CH₂—CH₂X, CH₂—CH₂—CH₂X, O—CH₂—CH₂—CH₂X (wherein X=F, Cl, Br or I), CN,(C═O)—R′, N(R′)₂, NO₂, (C═O)N(R′)₂, O(CO)R′, OR′, SR′, COOR′, R_(ph),CR′═CR′—R_(ph), CR₂′—CR₂′—R_(ph) (wherein R_(ph) represents anunsubstituted or substituted phenyl group with the phenyl substituentsbeing chosen from any of the non-phenyl substituents defined for R¹-R₁₀and wherein R′ is H or a lower alkyl group) and a tri-alkyl tin;

(vii) R⁵ is selected from the group consisting of H, F, Cl, Br, I, alower alkyl group, (CH₂)_(n)OR′ (wherein n=1, 2, or 3), CF₃, CH₂—CH₂X,O—CH₂—CH₂X, CH₂—CH₂—CH₂X, O—CH₂—CH₂—CH₂X (wherein X=F, Cl, Br or I), CN,(C═O)—R′, N(R′)₂, NO₂, (C═O)N(R′)₂, O(CO)R′, OR′, SR′, COOR′, R_(ph),CR′═CR′—R_(ph), CR₂′—CR₂′—R_(ph) (wherein R_(ph) represents anunsubstituted or substituted phenyl group with the phenyl substituentsbeing chosen from any of the non-phenyl substituents defined for R¹-R¹⁰and wherein R′ is H or a lower alkyl group) and a tri-alkyl tin;

(viii) R⁶ is selected from the group consisting of H, F, Cl, Br, I, alower alkyl group, (CH₂)_(n)OR′ (wherein n=1, 2, or 3), CF₃, CH₂—CH₂X,O—CH₂—CH₂X, CH₂—CH₂—CH₂X, O—CH₂—CH₂—CH₂X (wherein X=F, Cl, Br or I), CN,(C═O)—R′, N(R′)₂, NO₂, (C═O)N(R′)₂, O(CO)R′, OR′, SR′, COOR′, R_(ph),CR′═CR′—R_(ph), CR₂′—CR₂′—R_(ph) (wherein R_(ph) represents anunsubstituted or substituted phenyl group with the phenyl substituentsbeing chosen from any of the non-phenyl substituents defined for R¹-R¹⁰and wherein R′ is H or a lower alkyl group) and a tri-alkyl tin;

(ix) R⁷ is selected from the group consisting of H, F, Cl, Br, I, alower alkyl group, (CH₂)_(n)OR′ (wherein n=1, 2, or 3), CF₃, CH₂—CH₂X,O—CH₂—CH₂X, CH₂—CH₂—CH₂X, O—CH₂—CH₂—CH₂X (wherein X=F, Cl, Br or I), CN,(C═O)—R′, N(R′)₂, NO₂, (C═O)N(R′)₂, O(CO)R′, OR′, SR′, COOR′, R_(ph),CR′═CR′—R_(ph), CR₂′—CR₂′—R_(ph) (wherein R_(ph) represents anunsubstituted or substituted phenyl group with the phenyl substituentsbeing chosen from any of the non-phenyl substituents defined for R¹-R¹⁰and wherein R′ is H or a lower alkyl group) and a tri-alkyl tin;

(x) R⁸ is selected from the group consisting of H, F, Cl, Br, I, a loweralkyl group, (CH₂)_(n)OR′ (wherein n=^(˜)1, 2, or 3), CF₃, CH₂—CH₂X,O—CH₂—CH₂X, CH₂—CH₂—CH₂X, O—CH₂—CH₂—CH₂X (wherein X=F, Cl, Br or I), CN,(C═O)—R′, N(R′)₂, NO₂, (C═O)N(R′)₂, O(CO)R′, OR′, SR′, COOR′, R_(ph),CR′═CR′—R_(ph), CR₂′—CR₂′—R_(ph) (wherein R_(ph) represents anunsubstituted or substituted phenyl group with the phenyl substituentsbeing chosen from any of the non-phenyl substituents defined for R¹-R¹⁰and wherein R′ is H or a lower alkyl group) and a tri-alkyl tin;

(xi) R⁹ is selected from the group consisting of H, F, Cl, Br, I, alower alkyl group, (CH₂)_(n)OR′ (wherein n=1, 2, or 3), CF₃, CH₂—CH₂X,O—CH₂—CH₂X, CH₂—CH₂—CH₂X, O—CH₂—CH₂—CH₂X (wherein X=F, Cl, Br or I), CN,(C═O)—R′, N(R′)₂, NO₂, (C═O)N(R′)₂, O(CO)R′, OR′, SR′, COOR′, R_(ph),CR′═CR′—R_(ph), CR₂′—CR_(2′)—R_(ph) (wherein R_(ph) represents anunsubstituted or substituted phenyl group with the phenyl substituentsbeing chosen from any of the non-phenyl substituents defined for R¹-R¹⁰and wherein R′ is H or a lower alkyl group) and a tri-alkyl tin;

(xii) R¹⁰ is selected from the group consisting of H, F, Cl, Br, I, alower alkyl group, (CH₂)_(n)OR′ (wherein n=1, 2, or 3), CF₃, CH₂—CH₂X,O—CH₂—CH₂X, CH₂—CH₂—CH₂X, O—CH₂—CH₂—CH₂X (wherein X=F, Cl, Br or I), CN,(C═O)—R′, N(R′)₂, NO₂, (C═O)N(R′)₂, O(CO)R′, OR′, SR′, COOR′, R_(ph),CR₂′—CR₂′—R_(ph) (wherein R_(ph) represents an unsubstituted orsubstituted phenyl group with the phenyl substituents being chosen fromany of the non-phenyl substituents defined for R¹-R¹⁰ and wherein R′ isH or a lower alkyl group) and a tri-alkyl tin;

alternatively, one of R³-R¹⁰ may be a chelating group (with or without achelated metal group) of the form W-L or V—W-L, wherein V is selectedfrom the group consisting of —COO—, —CO—, —CH₂O— and —CH₂NH—; W is—(CH₂)_(n) where n=0, 1, 2, 3, 4, or 5; and L is:

wherein M is selected from the group consisting of Tc and Re;

and radiolabeled derivatives and pharmaceutically acceptable saltsthereof, where at least one of the substituent moieties comprises adetectable label;

(B) imaging said patient; then

(C) administering to said patient in need thereof at least oneanti-amyloid agent;

(D) subsequently administering to said patient in need thereof aneffective amount of a compound of formula (I);

(E) imaging said patient; and

(F) comparing levels of amyloid deposition in said patient beforetreatment with at least one anti-amyloid agent to levels of amyloiddeposition in said patient after treatment with at least oneanti-amyloid agent.

The detectable label includes any atom or moiety which can be detectedusing an imaging technique known to those skilled in the art. Typically,the detectable label is selected from the group consisting of ³H, ¹³¹I,¹²⁵I, ¹²³I, ⁷⁶Br, ⁷⁵Br, ¹⁸F, —CH₂—CH₂—X*, O—CH₂—CH₂—X*, CH₂—CH₂—CH₂—X*,O—CH₂—CH₂—CH₂—X* (wherein X*=¹³¹I, ¹²³I, ⁷⁶Br, ⁷⁵Br or ¹⁸F), ¹⁹F, ¹²⁵I,a carbon-containing substituent selected from the group consisting oflower alkyl, (CH2)nOR′, CF₃, CH₂—CH₂X, O—CH₂—CH₂X, CH₂—CH₂—CH₂X,O—CH₂—CH₂—CH₂X (wherein X=F, Cl, Br or I), CN, (C═O)—R′, (C═O)N(R′)₂,O(CO)R′, COOR′, CR′═CR′—R_(ph) and CR₂′—CR₂′—R_(ph) wherein at least onecarbon is ¹¹C, ¹³C or ¹⁴C and a chelating group (with chelated metalgroup) of the form W-L* or V—W-L*, wherein V is selected from the groupconsisting of —COO—, —CO—, —CH₂O— and —CH₂NH—; W is —(CH₂)_(n) wheren=0, 1, 2, 3, 4, or 5; and L* is:

wherein M* is ^(99m)Tc. In a preferred embodiment, the detectable labelis a radiolabel.

In a preferred embodiment, the detectable label is a radiolabel,

Amyloid Probes

The amyloid probe of the present invention is any compound of formula(I), described above.

In some embodiments, the amyloid probe is a compound of formula (II)

or a radiolabeled derivative, pharmaceutically acceptable salt, hydrate,solvate or prodrug of the compound, wherein:

R¹ is hydrogen, —OH, —NO₂, —CN, —COOR, —OCH₂OR, C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, C₁-C₆ alkoxy or halo;

R is C₁-C₆ alkyl;

R² is hydrogen or halo;

R³ is hydrogen, C₁-C₆ alkyl, C₂-C₆ alkenyl or C₂-C₆ alkynyl; and

R⁴ is hydrogen, C₁-C₆ alkyl, C₂-C₆ alkenyl or C₂-C₆ alkynyl, wherein thealkyl, alkenyl or alkynyl comprises a radioactive carbon or issubstituted with a radioactive halo when R² is hydrogen or anon-radioactive halo;

provided that when R¹ is hydrogen or —OH, R² is hydrogen and R⁴ is—¹¹CH₃, then R³ is C₂-C₆ alkyl, C₂-C₆ alkenyl or C₂-C₆ alkynyl; and

further provided that when R¹ is hydrogen, R² hydrogen and R⁴ is —(CH₂)₃¹⁸F, then R³ is C₂-C₆ alkyl, C₂-C₆ alkenyl or C₂-C₆ alkynyl, where atleast one of the substituent moieties comprises a detectable label.

In one embodiment, R² in the compounds of formula (II) contains aradioactive halo.

“Alkyl” refers to a saturated straight or branched chain hydrocarbonradical. Examples include without limitation methyl, ethyl, propyl,iso-propyl, butyl, iso-butyl, tert-butyl, n-pentyl and n-hexyl. The term“lower alkyl” refers to C₁-C₆ alkyl.

“Alkenyl” refers to an unsaturated straight or branched chainhydrocarbon radical comprising at least one carbon to carbon doublebond. Examples include without limitation ethenyl, propenyl,iso-propenyl, butenyl, iso-butenyl, text-butenyl, n-pentenyl andn-hexenyl.

“Alkynyl” refers to an unsaturated straight or branched chainhydrocarbon radical comprising at least one carbon to carbon triplebond. Examples include without limitation ethynyl, propynyl,iso-propynyl, butynyl, iso-butynyl, tert-butynyl, pentynyl and hexynyl.

“Alkoxy” refers to an alkyl group bonded through an oxygen linkage.

“Halo” refers to a fluoro, chloro, bromo or iodo radical.

“Radioactive halo” refers to a radioactive halo, i.e. radiofluoro,radiochloro, radiobromo or radioiodo.

In another embodiment, the thioflavin compound of formula (I) isselected from the group consisting of structures 1-45 or a radiolabeledderivative thereof:

In preferred embodiments, the amyloid probe is{N-methyl-¹¹C}2-[4′-(methylamino)phenyl]6-hydroxybenzothiazole(“[¹¹C]PIB”) or{N-methyl-³H}2-[4′-(methylamino)phenyl]6-hydroxybenzothiazole(“[³H]PIB”).

“Effective amount” refers to the amount required to produce a desiredeffect. Examples of an “effective amount” include amounts that enabledetecting and imaging of amyloid deposit(s) in vivo or in vitro, thatyield acceptable toxicity and bioavailability levels for pharmaceuticaluse, and/or prevent cell degeneration and toxicity associated withfibril formation.

Compounds of formulas (I) and (II), also referred to herein as“thioflavin compounds,” “thioflavin derivatives,” or “amyloid probes,”have each of the following characteristics: (1) specific binding tosynthetic Aβ in vitro and (2) ability to cross a non-compromised bloodbrain barrier in vivo.

The thioflavin compounds and radiolabeled derivatives thereof offormulas (I) and (II) and structures 1-45 cross the blood brain barrierin vivo and bind to Aβ deposited in neuritic (but not diffuse) plaques,to Aβ deposited in cerebrovascular amyloid, and to the amyloidconsisting of the protein deposited in NFT. The present compounds arenon-quaternary amine derivatives of Thioflavin S and T which are knownto stain amyloid in tissue sections and bind to synthetic Aβ in vitro.Kelenyi J. Histochem. Cytochem. 15: 172 (1967); Burns et al. J. Path.Bact. 94:337 (1967); Guntem et al. Experientia 48: 8 (1992); LeVineMeth. Enzymol. 309: 274 (1999).

The method of this invention determines the presence and location ofamyloid deposits in an organ or body area, preferably brain, of apatient. The present method comprises administration of a detectablequantity of an amyloid probe of formulas (I) or (II) and structures1-45. In some embodiments, the amyloid probe is chosen from structures1-45, as shown above. An amyloid probe may be administered to a patientas a pharmaceutical composition or a pharmaceutically acceptablewater-soluble salt thereof.

“Pharmaceutically acceptable salt” refers to an acid or base salt of theinventive compound, which salt possesses the desired pharmacologicalactivity and is neither biologically nor otherwise undesirable. The saltcan be formed with acids that include without limitation acetate,adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfatebutyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate,glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloridehydrobromide, hydroiodide, 2-hydroxyethane-sulfonate, lactate, maleate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate,thiocyanate, tosylate and undecanoate. Examples of a base salt includewithout limitation ammonium salts, alkali metal salts such as sodium andpotassium salts, alkaline earth metal salts such as calcium andmagnesium salts, salts with organic bases such as dicyclohexylaminesalts, N-methyl-D-glucamine, and salts with amino acids such as arginineand lysine. In some embodiments, the basic nitrogen-containing groupscan be quarternized with agents including lower alkyl halides such asmethyl, ethyl, propyl and butyl chlorides, bromides and iodides; dialkylsulfates such as dimethyl, diethyl, dibutyl and diamyl sulfates; longchain halides such as decyl, lauryl, myristyl and stearyl chlorides,bromides and iodides; and aralkyl halides such as phenethyl bromides.

Generally, the dosage of the detectably labeled thioflavin derivativewill vary depending on considerations such as age, condition, sex, andextent of disease in the patient, contraindications, if any, concomitanttherapies and other variables, to be adjusted by a physician skilled inthe art. Dosage can vary from 0.001 μg/kg to 10 μg/kg, preferably 0.01μg/kg to 1.0 μg/kg.

Administration to the subject may be local or systemic and accomplishedintravenously, intraarterially, intrathecally (via the spinal fluid) orthe like. Administration may also be intradermal or intracavitary,depending upon the body site under examination. After a sufficient timehas elapsed for the compound to bind with the amyloid, for example 30minutes to 48 hours, the area of the subject under investigation isexamined by routine imaging techniques such as MRS/MRI, SPECT, planarscintillation imaging, PET, and any emerging imaging techniques, aswell. The exact protocol will necessarily vary depending upon factorsspecific to the patient, as noted above, and depending upon the bodysite under examination, method of administration and type of label used;the determination of specific procedures would be routine to the skilledartisan. For brain imaging, preferably, the amount (total or specificbinding) of the bound radioactively labeled thioflavin derivative oranalogue of the present invention is measured and compared (as a ratio)with the amount of labeled thioflavin derivative bound to the cerebellumof the patient. This ratio is then compared to the same ratio inage-matched normal brain.

The amyloid probes of the present invention are advantageouslyadministered in the form of injectable compositions, but may also beformulated into well known drug delivery systems (e.g., oral, rectal,parenteral (intravenous, intramuscular, or subcutaneous),intracisternal, intravaginal, intraperitoneal, local (powders, ointmentsor drops), or as a buccal or nasal spray). A typical composition forsuch purpose comprises a pharmaceutically acceptable carrier. Forinstance, the composition may contain about 10 mg of human serum albuminand from about 0.5 to 500 micrograms of the labeled thioflavinderivative per milliliter of phosphate buffer containing NaCl. Otherpharmaceutically acceptable carriers include aqueous solutions,non-toxic excipients, including salts, preservatives, buffers and thelike, as described, for instance, in REMINGTON′S PHARMACEUTICALSCIENCES, 15th Ed. Easton: Mack Publishing Co. pp. 1405-1412 and1461-1487 (1975) and THE NATIONAL FORMULARY XIV., 14th Ed. Washington:American Pharmaceutical Association (1975), the contents of which arehereby incorporated by reference.

Particularly preferred amyloid probes of the present invention are thosethat, in addition to specifically binding amyloid in vivo and capable ofcrossing the blood brain barrier, are also non-toxic at appropriatedosage levels and have a satisfactory duration of effect.

According to the present invention, a pharmaceutical compositioncomprising an amyloid probe of formula (I) or formula (II) or one of thestructures 1-45, is administered to subjects in whom amyloid or amyloidfibril formation are anticipated, e.g., patients clinically diagnosedwith Alzheimer's disease or another disease associated with amyloiddeposition.

Examples of non-aqueous solvents are propylene glycol, polyethyleneglycol, vegetable oil and injectable organic esters such as ethyloleate. Aqueous carriers include water, alcoholic/aqueous solutions,saline solutions, parenteral vehicles such as sodium chloride, Ringer'sdextrose, etc. Intravenous vehicles include fluid and nutrientreplenishers. Preservatives include antimicrobial, anti-oxidants,chelating agents and inert gases. The pH and exact concentration of thevarious components the pharmaceutical composition are adjusted accordingto routine skills in the art. See, Goodman and Gilman's THEPHARMACOLOGICAL BASIS FOR THERAPEUTICS (7th Ed.).

Imaging

The invention employs amyloid probes which, in conjunction withnon-invasive neuroimaging techniques such as magnetic resonancespectroscopy (MRS) or imaging (MRI), or gamma imaging such as positronemission tomography (PET) or single-photon emission computed tomography(SPECT), are used to quantify amyloid deposition in vivo. The methodinvolves imaging a patient to establish a baseline of amyloiddeposition. The term “baseline” refers to the amount and distribution ofa patient's amyloid deposition prior to initiation of the anti-amyloidtherapy. The method further involves at least one imaging session of apatient following administration of an anti-amyloid therapy. The presentmethod may involve imaging a patient before and after treatment with atleast one anti-amyloid agent. Imaging may be performed at any timeduring the treatment.

The term “in vivo imaging” refers to any method which permits thedetection of a labeled thioflavin derivative of formulas (I) or (II) orone of structures 1-45. For gamma imaging, the radiation emitted fromthe organ or area being examined is measured and expressed either astotal binding or as a ratio in which total binding in one tissue isnormalized to (for example, divided by) the total binding in anothertissue of the same subject during the same in vivo imaging procedure.Total binding in vivo is defined as the entire signal detected in atissue by an in vivo imaging technique without the need for correctionby a second injection of an identical quantity of labeled compound alongwith a large excess of unlabeled, but otherwise chemically identicalcompound. A “subject” is a mammal, preferably a human, and mostpreferably a human suspected of having a disease associated with amyloiddeposition, such as AD and/or dementia. The term “subject” and “patient”are used interchangeably herein.

For purposes of in vivo imaging, the type of detection instrumentavailable is a major factor in selecting a given label. For instance,radioactive isotopes and ¹⁸F are particularly suitable for in vivoimaging in the methods of the present invention. The type of instrumentused will guide the selection of the radionuclide or stable isotope. Forinstance, the radionuclide chosen must have a type of decay detectableby a given type of instrument. Another consideration relates to thehalf-life of the radionuclide. The half-life should be long enough sothat it is still detectable at the time of maximum uptake by the target,but short enough so that the host does not sustain deleteriousradiation. The radiolabeled compounds of the invention can be detectedusing gamma imaging wherein emitted gamma irradiation of the appropriatewavelength is detected. Methods of gamma imaging include, but are notlimited to, SPECT and PET. Preferably, for SPECT detection, the chosenradiolabel will lack a particulate emission, but will produce a largenumber of photons in a 140-200 keV range. For PET detection, theradiolabel will be a positron-emitting radionuclide such as ¹⁸F whichwill annihilate to form two 511 keV gamma rays which will be detected bythe PET camera.

In the present invention, amyloid binding compounds/probes, which areuseful for in vivo imaging and quantification of amyloid deposition, areadministered to a patient. These compounds are to be used in conjunctionwith non-invasive neuroimaging techniques such as magnetic resonancespectroscopy (MRS) or imaging (MRI), positron emission tomography (PET),and single-photon emission computed tomography (SPECT). In accordancewith this invention, the thioflavin derivatives may be labeled with ¹⁸For ¹³C for MRS/MRI by general organic chemistry techniques known to theart. See, e.g., March, J. ADVANCED ORGANIC CHEMISTRY: REACTIONS,MECHANISMS, AND STRUCTURE (3rd Edition, 1985), the contents of which arehereby incorporated by reference. The thioflavin derivatives also may beradiolabeled with ¹⁸F, ¹¹C, ⁷⁵Br, or ⁷⁶Br for PET by techniques wellknown in the art and are described by Fowler, J. and Wolf, A. inPOSITRON EMISSION TOMOGRAPHY AND AUTORADIOGRAPHY (Phelps, M., Mazziota,J., and Schelbert, H. eds.) 391-450 (Raven Press, NY 1986) the contentsof which are hereby incorporated by reference. The thioflavinderivatives also may be radiolabeled with ¹²³I for SPECT by any ofseveral techniques known to the art. See, e.g., Kulkarni, Int. J. Rad.Appl. & Inst. (Part B) 18: 647 (1991), the contents of which are herebyincorporated by reference. In addition, the thioflavin derivatives maybe labeled with any suitable radioactive iodine isotope, such as, butnot limited to ¹³¹I, ¹²⁵I, or ¹²³I, by iodination of a diazotized aminoderivative directly via a diazonium iodide, see Greenbaum, F. Am. J.Pharm. 108: 17 (1936), or by conversion of the unstable diazotized amineto the stable triazene, or by conversion of a non-radioactivehalogenated precursor to a stable tri-alkyl tin derivative which thencan be converted to the iodo compound by several methods well known tothe art. See, Satyamurthy and Barrio J. Org. Chem. 48: 4394 (1983),Goodman et al., J. Org. Chem. 49: 2322 (1984), and Mathis et al., J.Labell. Comp. and Radiopharm. 1994: 905; Chumpradit et al., J. Med.Chem. 34: 877 (1991); Zhuang et al., J. Med. Chem. 37: 1406 (1994);Chumpradit et al., J. Med. Chem. 37: 4245 (1994). For example, a stabletriazene or tri-alkyl tin derivative of thioflavin or its analogues isreacted with a halogenating agent containing ¹³¹I, ¹²⁵I, ¹²³I, ⁷⁶Br,⁷⁵Br, ¹⁸F or ¹⁹F. Thus, the stable tri-alkyl tin derivatives ofthioflavin and its analogues are novel precursors useful for thesynthesis of many of the radiolabeled compounds within the presentinvention. As such, these tri-alkyl tin derivatives are one embodimentof this invention.

The thioflavin derivatives also may be radiolabeled with known metalradiolabels, such as Technetium-99m (^(99m)Tc). Modification of thesubstituents to introduce ligands that bind such metal ions can beeffected without undue experimentation by one of ordinary skill in theradiolabeling art. The metal radiolabeled thioflavin derivative can thenbe used to detect amyloid deposits. Preparing radiolabeled derivativesof Tc^(99m) is well known in the art. See, for example, Zhuang et al.,“Neutral and stereospecific Tc-99m complexes: [99mTc]N-benzyl-3,4-di-(N-2-mercaptoethyl)-amino-pyrrolidines (P-BAT)”Nuclear Medicine & Biology 26(2):217-24, (1999); Oya et al., “Small andneutral Tc(v)O BAT, bisaminoethanethiol (N2S2) complexes for developingnew brain imaging agents” Nuclear Medicine & Biology 25(2):135-40,(1998); and Horn et al., “Technetium-99m-labeled receptor-specificsmall-molecule radiopharmaceuticals: recent developments and encouragingresults” Nuclear Medicine & Biology 24(6):485-98, (1997).

The methods of the present invention may use isotopes detectable bynuclear magnetic resonance spectroscopy for purposes of in vivo imagingand spectroscopy. Elements particularly useful in magnetic resonancespectroscopy include ¹⁸F and ¹³C.

Suitable radioisotopes for purposes of this invention includebeta-emitters, gamma-emitters, positron-emitters, and x-ray emitters.These radioisotopes include ¹³¹I, ¹²³I, ¹⁸F, ¹¹C, ⁷⁵Br, and ⁷⁶Br.Suitable stable isotopes for use in Magnetic Resonance Imaging (MRI) orSpectroscopy (MRS), according to this invention, include ¹⁸F and ¹³C.Suitable radioisotopes for in vitro quantification of amyloid inhomogenates of biopsy or post-mortem tissue include ¹²⁵I, ¹⁴C, and ³H.The preferred radiolabels are ¹¹C or ¹⁸F for use in PET in vivo imaging,¹²³I for use in SPECT imaging, ¹⁹F for MRS/MRI, and ³H or ¹⁴C for invitro studies. However, any conventional method for visualizingdiagnostic probes can be utilized in accordance with this invention.

According to an aspect of the invention which relates to a method ofdetecting amyloid deposits in biopsy tissue, the method involvesincubating formalin-fixed tissue with a solution of a thioflavin amyloidbinding compound chosen from compounds of formulas (I) and (II) orstructures 1-45, described above. Preferably, the solution is 25-100%ethanol, (with the remainder being water) saturated with a thioflavinamyloid binding compound of formulas (I) or (II) or structures 1-45according to the invention. Upon incubation, the compound stains orlabels the amyloid deposit in the tissue, and the stained or labeleddeposit can be detected or visualized by any standard method. Suchdetection means include microscopic techniques such as bright-field,fluorescence, laser-confocal and cross-polarization microscopy.

The method of quantifying the amount of amyloid in biopsy tissueinvolves incubating a labeled derivative of thioflavin according to thepresent invention, or a water-soluble, non-toxic salt thereof, withhomogenate of biopsy or post-mortem tissue. The tissue is obtained andhomogenized by methods well known in the art. The preferred label is aradiolabel, although other labels such as enzymes, chemiluminescent andimmunofluorescent compounds are well known to skilled artisans. Thepreferred radiolabel is ¹²⁵I, ¹⁴C, ³H which is contained in asubstituent substituted on one of the compounds of formulas (I) or (II)or structures 1-45. Tissue containing amyloid deposits will bind to thelabeled derivatives of the thioflavin amyloid binding compounds of thepresent invention. The bound tissue is then separated from the unboundtissue by any mechanism known to the skilled artisan, such as filtering.The bound tissue can then be quantified through any means known to theskilled artisan. The units of tissue-bound radiolabeled thioflavinderivative are then converted to units of micrograms of amyloid per 100mg of tissue by comparison to a standard curve generated by incubatingknown amounts of amyloid with the radiolabeled thioflavin derivative.

The ability of the compound of formulas (I) and (II) or structures 1-45to specifically bind to amyloid plaques over neurofibrially tangles isparticularly true at concentrations less than 10 nM, which includes thein vivo concentration range of PET radiotraces. At these lowconcentrations, in homogenates of brain tissue which contain onlytangles and no plaques, significant binding does not result whencompared to control brain tissue containing neither plaques nor tangles.However, incubation of homogenates of brain tissue which contains mainlyplaques and some tangles with radiolabeled compounds of Formula (I) or(II) or structures 1-45, results in a significant increase in bindingwhen compared to control tissue without plaques or tangles. This datasuggests the advantage that these compounds are specific for Aβ depositsat concentrations less than 10 nM. These low concentrations are thendetectable with PET studies, making PET detection using radiolabeledcompounds of Formula (I) or Formula (II) or structures 1-45 which arespecific for Aβ deposits possible. The use of such compounds permits PETdetection in Aβ deposits such as those found in plaques andcerebrovascular amyloid. Since it has been reported that Aβ levels inthe frontal cortex are increased prior to tangle formation, this wouldsuggest that radiolabeled compounds of Formula (I) or Formula (II) orstructures 1-45, used as PET tracers, would be specific for the earliestchanges in AD cortex. Naslund et al. JAMA 283:1571 (2000).

Anti-Amyloid Therapies

The present method for determining the efficacy of therapy in thetreatment of amyloidosis involves administering to a patient in needthereof a compound of formulas (I) or (II) or structure 1-45 and imagingthe patient, and, after said imaging, administering at least oneanti-amyloid agent/anti-amyloid therapy said patient. The amountadministered, the route of administration, and the duration of therapyare determined by one skilled in the art based on age, weight, andcondition of the patient. Such determinations are within the purview ofthe skilled practitioner. Suitable amounts include, but are not limitedto, 0.01 to 100 mg/kg. Suitable routes of administration include, butare not limited to oral, subcutaneous and intravenous. Suitabledurations of therapy include, but are not limited to one single dose tofour doses per day given indefinitely. Suitable times to image include,but are not limited to immediately after the first dose to ten yearsafter the most recent dose. Preferred times to image would be between 7days and 6 months after the most recent dose.

An “Anti-amyloid agent” or an “anti-amyloid therapy” is any agent orcombination of agents that treat or prevent amyloidosis. Examples ofdiseases associated with amyloid deposition, amyloidosis, includeAlzheimer's Disease, Down's Syndrome, Type 2 diabetes mellitus,hereditary cerebral hemorrhage amyloidosis (Dutch), amyloid A(reactive), secondary amyloidosis, MCI, familial mediterranean fever,familial amyloid nephropathy with urticaria and deafness (Muckle-wellsSyndrome), amyloid lambda L-chain or amyloid kappa L-chain (idiopathic,myeloma or macroglobulinemia-associated) A beta 2M (chronichemodialysis), ATTR (familial amyloid polyneuropathy (Portuguese,Japanese, Swedish)), familial amyloid cardiomyopathy (Danish), isolatedcardiac amyloid, systemic senile amyloidoses, AIAPP or amylininsulinoma, atrial naturetic factor (isolated atrial amyloid),procalcitonin (medullary carcinoma of the thyroid), gelsolin (familialamyloidosis (Finnish)), cystatin C (hereditary cerebral hemorrhage withamyloidosis (Icelandic)), AApo-A-I (familial amyloidoticpolyneuropathy-Iowa), AApo-A-II (accelerated senescence in mice),fibrinogen-associated amyloid; and Asor or Pr P-27 (scrapie, CreutzfeldJacob disease, Gertsmann-Straussler-Scheinker syndrome, bovinespongiform encephalitis) or in cases of persons who are homozygous forthe apolipoprotein E4 allele, and the condition associated withhomozygosity for the apolipoprotein E4 allele or Huntington's disease.The invention encompasses diseases associated with amyloid plaquedeposition. Preferably, the disease associated with amyloid depositionis AD.

The term “therapy” includes treating and/or preventing disease.

“Treating” refers to:

(i) preventing a disease, disorder or condition from occurring in ananimal that may be predisposed to the disease, disorder and/or conditionbut has not yet been diagnosed as having it;

(ii) inhibiting the disease, disorder or condition, i.e., arresting itsdevelopment; and/or

(iii) relieving the disease, disorder or condition, i.e., causingregression of the disease, disorder and/or condition.

The term “treating” or “treatment” does not necessarily mean total cure.Any alleviation of any undesired symptom or pathological effect of thedisease to any extent or the slowing down of the progress of the diseasecan be considered treatment. Furthermore, treatment may include actswhich may worsen the patient's overall feeling of well being orappearance. For example, the administration of chemotherapy in cancerpatients which may leave the patients feeling “sicker” is stillconsidered treatment.

The term “preventing” refers to decreasing the probability that anorganism contracts or develops a disease associated with amyloiddeposition. The term “preventing” preferably refers to reducing thepercentage of individuals who develop the disease relative to a controlgroup that does not undergo administration of an anti-amyloid agent.

The present invention is directed to amyloid imaging serving as asurrogate marker of efficacy for anti-amyloid therapy. Administration ofan amyloid probe to establish a baseline of amyloid deposition andsubsequent imaging of a patient both before and after treatment of thepatient with an anti-amyloid agent allows for determination of theefficacy of the anti-amyloid therapy. The present method can be used todetermine the efficacy of any anti-amyloid treatment because an amyloidprobe can be administered, and the patient can be imaged, before andafter any anti-amyloid therapy. The present method contemplatesdetermining anti-amyloid therapies which are ineffective for treatingdiseases associated with amyloid deposition, as well as anti-amyloidtherapies which are effective for treating diseases associated withamyloid deposition. A person of ordinary skill in the art can determinethe conditions and dosing of the anti-amyloid therapy according toappropriate protocols. Therefore, the present invention contemplatesdetermining the efficacy of anti-amyloid therapies that are now known,as well as therapies that are yet to be discovered. Exemplarynon-limiting anti-amyloid therapies are described below.

In some embodiments, the efficacy of acetylcholinesterase inhibitors inthe treatment of amyloidosis is determined by the present method.Acetylcholinesterase therapy is based on studies of degenerationpatterns in AD which identified substantial decreases among groups ofneurons in the basal forebrain. These cells all used the transmitteracetylcholine, and their loss meant that less acetylcholine was beingreleased at their former terminals in the cortex. Several drugs, such astacrine, donepezil, rivastigmine and galantamine have been developedbased on these findings, and are hypothesized to work by inhibiting theenzyme acetylcholinesterase (Ingram, V., American Scientist, 2003,91(4):312-321).

In other embodiments, the efficacy of anti-amyloid therapy targetingenzymes responsible for formation of noxious fragments of amyloidprecursor protein (APP) in the treatment of amyloidosis is determined bythe present method. In some embodiments, the noxious fragments of theamyloid precursor protein (APP) is misfolded Aβ peptide. For example,the overproduction of Aβ1-42 fragment is considered by some scientiststo be a root cause of AD. The Aβ1-42 fragment is formed by cleavage ofAPP by the β-secretase enzyme (BACE1) (which produces the aminoterminus) and the γ-secretase enzyme (which cleaves the carboxylterminus of APP). Inhibitors of these secretase enzymes may be used asanti-amyloid therapies (Ingram, V., American Scientist, 2003,91(4):312-321).

In some embodiments, the efficacy of immunotherapeutic strategies in thetreatment of amyloidosis can be determined by the present method.Immunotherapy works by using the patient's immune system to locate anddestroy amyloid plaques and many immunotherapy strategies are beingactively pursued by scientists. The immunotherapeutic strategies can beeither passive or active. For example, in active immunotherapy, apatient may receive an injection or nasal-spray application of the Aβpeptide, leading to an anti-amyloid immune response. Passiveimmunotherapy, on the other hand, might involve bypassing the betaamyloid protein, using instead antiserum that has already been producedin response to beta amyloid. Immunotherapy, involving antibodies againstAβ peptide, has been studied for the treatment of AD. For example,AN-1792 is a preparation of preaggregated synthetic amyloid-beta (Aβ;1-42 length) along with QS-21 adjuvant (Hock, C. et al., 2003, Neuron,38:547-554). Approximately 300 AD patients have been treated with thispreparation prior to suspension of the clinical trial due to sideeffects (Birmingham, K. and Frantz, S., 2002, Nature Medicine,8:199-200).

In other embodiments, the efficacy of neuroprotective strategies in thetreatment of amyloidosis is determined by the present method. Forexample, many clinicians recommend that patients take high doses(1000-2000 IU/day) of vitamin E. Other types of neuroprotectivestrategies that have been suggested for the treatment of amyloidosis arehigh doses of vitamin C, calcium channel modulators, free-radicalscavengers, and metal ion chelators (Selkoe, et al., Annu. Rev.Pharmacol. Toxicol., 2003, 43:545-84).

In some embodiments, the efficacy of anti-inflammatory drugs (NSAIDs)strategies in the treatment of amyloidosis is determined by the presentmethod. Treatments involving NSAIDs are based on evidence that acellular inflammatory response in the cortex is elicited by theprogressive accumulation of Aβ peptide. Exemplary anti-inflammatorydrugs are prednisone, nonspecific cyclooxygenase inhibitors, andcyclooxygenase-2 inhibitors. (Clark, M., et al., Annals of InternalMedicine, 2003, 138(5):400-410; and Hardy, John, Annu. Rev. Med., 2004,55:15-25).

In some embodiments, the efficacy of cholesterol-lowering therapiesincluding, but not limited to, the 3-hydroxy-3-methylglutaryl coenzyme Areductase inhibitors (statins) is determined by the present method.Treatments involving cholesterol-lowering drugs (such as statins) arebased on epidemiological evidence that patients treated with statinshave a lower incidence of AD and that statins can alter the metabolismof Aβ to decrease Aβ levels (Wolozin, B (2002) Cholesterol andAlzheimer's disease. Biochemical Society Transactions. 30:525-529).Exemplary cholesterol-lowering statin drugs include lovastatin,pravastatin, rosuvastatin, fluvastatin, atorvastatin and simvastatin.Other cholesterol-lowering drugs include niacin, cholestyramine,fenofibrate, colesevelam and ezetimibe.

In other embodiments, the efficacy of small molecules that eliminate theneurotoxicity of the aggregated Aβ1-42 in the treatment of amyloidosisis determined by the present method. Such a drug, preferablyadministered early in disease progression, would “detoxify” thegradually accumulating Aβ peptide before any permanent damage isinflicted on the neurons. (Clark, M., et al., Annals of InternalMedicine, 2003, 138(5):400-410)

In some embodiments, the efficacy of “decoy peptides” in the treatmentof amyloidosis is determined by the present method. Decoy peptides aresmall molecules that bind to the aggregating Aβ1-42 peptide and force itto assume a nontoxic structure. Exemplary decoy peptides are smallpeptides (5, 6 or 9 amino acids long), selected from large libraries ofprotein fragments by their ability to form a tight association withtagged Aβ1-42. (Clark, M., et al., Annals of Internal Medicine, 2003,138(5):400-410).

In other embodiments, the efficacy of cholesterol homeostasis modulationin the treatment of amyloidosis is determined by the present method.Chronic use of cholesterol-lowering drugs has recently been associatedwith a lower incidence of AD. Concurrently, high-cholesterol diets havebeen shown to increase Aβ pathology in animals, and cholesterol-loweringdrugs have been shown to reduce pathology in APP transgenic mice.Clinical trials are underway to study the effect of cholesterolhomeostasis modulation in the treatment of AD. (Hardy, John, Annu. Rev.Med., 2004, 55:15-25)

Certain antibodies such as the one termed m266 (DeMattos, R B, Bales, KR, Cummins, D J, Dodart, J C, Paul, S M, Holtzman, D M (2001)“Peripheral anti-A beta antibody alters CNS and plasma A beta clearanceand decreases brain A beta burden in a mouse model of Alzheimer'sdisease.” Proc. Natl. Acad. Sci. USA 98:8850-8855) or molecules otherthan antibodies (Matsuoka, Y, Saito, M, LaFrancois, J, Saito, M, Gaynor,K, Olm, V, Wang, L, Casey, E, Lu, Y, Shiratori, C, Lernere, C, Duff, K(2001) “Novel therapeutic approach for the treatment of Alzheimer'sdisease by peripheral administration of agents with an affinity tobeta-amyloid.” Journal of Neuroscience. 23:29-33) are believed to lowerbrain amyloid by binding to Aβ peptides in the blood, thereby creating a“peripheral sink” and shifting the equilibrium of Aβ from the brain tothe blood, where it can be cleared from the body. Such agents arereferred to herein as “peripheral sink agents.”

Evaluating the Efficacy of the Anti-Amyloid Therapy

The present method for determining the efficacy of therapy in thetreatment of amyloidosis involves administering to a patient in needthereof a compound of formulas (I) or (II) or structure 1-45 and imagingthe patient. After said imaging, at least one anti-amyloid agent isadministered to said patient. Then, an effective amount of a compound offormulas (I) or (II) or structure 1-45 is administered to the patientand the patient is imaged again. Finally, baseline levels of amyloiddeposition in the patient before treatment with the anti-amyloid agentare compared with levels of amyloid deposition in the patient followingtreatment with the anti-amyloid agent. Such a comparison is within thepreview of a skilled practitioner.

In some embodiments, the levels of amyloid deposition in the patientbefore treatment with the anti-amyloid agent will be higher than thelevels of amyloid deposition in the patient following treatment with theanti-amyloid agent. Such a result indicates that the anti-amyloidagent/anti-amyloid therapy is effective in the treatment of diseasesassociated with amyloid deposition.

For example, AN-1792 is a preparation of preaggregated syntheticamyloid-beta (Aβ; 1-42 length) along with QS-21 adjuvant. Approximately300 AD patients have been treated with this preparation prior tosuspension of the clinical trial due to side effects (Birmingham, K. andFrantz, S., 2002, Nature Medicine, 8:199-200). Despite this set back,optimism over this approach has been raised by two findings. First, inthe only autopsy report yet published regarding an AN-1792-treated ADpatient, there were several unusual findings including: (i) extensiveareas of neocortex with very few Aβ plaques; (ii) areas of cortex thatwere devoid of Aβ plaques contained densities of tangles, neuropilthreads and cerebral amyloid angiopathy (CAA) similar to unimmunized AD,but lacked plaque-associated dystrophic neurites and astrocyte clusters;(iii) in some regions devoid of plaques, Aβ-immunoreactivity wasassociated with microglia (Nicoll, J. et al., 2003, Nature Medicine,9:448-452). Second, in a small subset of 30 AN-1792-treated patients,those patients who generated antibodies against Aβ, as determined by atissue amyloid plaque immunoreactivity (TAPIR) assay showedsignificantly slower rates of decline of cognitive functions andactivities of daily living, as indicated by the Mini Mental StateExamination, the Disability Assessment for Dementia, and the VisualPaired Associates Test of delayed recall from the Wechsler Memory Scale,as compared to patients without such antibodies (Hock, C. et al., 2003,Neuron, 38:547-554).

It has been shown previously, that the benzothiazole amyloid-imaging PETtracer {N-methyl-¹¹C}2-[4′-(methylamino)phenyl]6-hydroxybenzothiazole([¹¹C]PIB) shows a clear difference in retention between AD patients andcontrol subjects, and that [¹¹C]PIB retention follows the knowntopography of amyloid deposition in AD brain (Klunk, et al., 2004, Ann.Neurol., 55(3):306-19). To determine whether benzothiazole amyloidimaging probes might be sensitive to changes in brain amyloid depositioncaused by an anti-amyloid therapy in general, testing by immunizationwith AN-1792 was performed. Studies were performed for the binding of{N-methyl-³H}2-[4′-(methylamino)phenyl]6-hydroxy-benzothiazole ([³H]PIB)to homogenates of frontal cortex and cerebellum obtained from controlsubjects (n=4), AD patients (n=5) and from a single AN-1792-treated ADcase (in duplicate). See, e.g., Example 9. The frontal cortex of ADpatients showed elevated [³H]PIB binding compared to control brain.However, [³H]PIB binding to the AN-1792-treated brain showed no increasein [³H]PIB binding over control frontal cortex. Taken together, thesedata suggest that benzothiazole amyloid imaging probes which are usefulas PET tracers, such as [¹¹C]PIB, could detect changes in amyloiddeposition in AD brain induced by AN-1792 treatment and by othertherapies that have a significant effect on brain amyloid deposition inAD.

Unless the context clearly dictates otherwise, the definitions ofsingular terms may be extrapolated to apply to their plural counterpartsas they appear in the application; likewise, the definitions of pluralterms may be extrapolated to apply to their singular counterparts asthey appear in the application.

The following examples are given to illustrate the present invention. Itshould be understood, however, that the invention is not to be limitedto the specific conditions or details described in these examples.Throughout the specification, any and all references to a publiclyavailable document, including U.S. patents, are specificallyincorporated into this patent application by reference.

EXAMPLES

Compounds of formulas (I) and (II), and the formulae of structures 1-45,can be prepared by methods that are well known in the art. See, e.g., WO02/16333 and U.S. Patent Publication No. 2003/0236391, published Dec.25, 2003, the entire contents of which are herein incorporated byreference.

All of the reagents used in the synthesis were purchased from AldrichChemical Company and used without further purification, unless otherwiseindicated. Melting points were determined on MeI-TEMP II and wereuncorrected. The ¹H NMR spectra of all compounds were measured on Bruker300 using TMS as internal reference and were in agreement with theassigned structures. The TLC was performed using Silica Gel 60 F₂₅₄ fromEM Sciences and detected under UV lamp. Flash chromatography wasperformed on silica gel 60 (230-400 mesh. Purchased from MallinckrodtCompany. The reverse phase TLC were purchased from Whiteman Company.

General Methodology for Synthesis of Compound of Formula (I):

R¹ is hydrogen, —OH, —NO₂, —CN, —COOR, —OCH₂OR, C₁-C₆ alkyl, C₂-C₆alkenyl, C₂-C₆ alkynyl, C₁-C₆ alkoxy or halo, wherein one or more of theatoms of R¹ may be a radiolabeled atom;

R is C₁-C₆ alkyl, wherein one or more of the carbon atoms may be aradiolabeled atom;

is hydrolysed by one of the following two procedures:

Preparation of 2-aminothiophenol via Hydrolysis

The 6-substituted 2-aminobenzothiazole (172 mmol) is suspended in 50%KOH (180 g KOH dissolved in 180 mL water) and ethylene glycol (40 mL).The suspension is heated to reflux for 48 hours. Upon cooling to roomtemperature, toluene (300 mL) is added and the reaction mixture isneutralized with acetic acid (180 mL). The organic layer is separatedand the aqueous layer is extracted with another 200 mL of toluene. Thetoluene layers are combined and washed with water and dried over MgSO₄.Evaporation of the solvent gives the desired product.

Preparation of 2-aminothiophenol via Hydrazinolysis

The 6-substituted -benzothiazole (6.7 mmol) is suspended in ethanol (11mL, anhydrous) and hydrazine (2.4 mL) is added under a nitrogenatmosphere at room temperature. The reaction mixture is heated to refluxfor 1 hour. The solvent is evaporated and the residue is dissolved intowater (10 mL) and adjusted to a pH of 5 with acetic acid. Theprecipitate is collected with filtration and washed with water to givethe desired product.

The resulting 5-substituted-2-amino-1-thiophenol of the form

is coupled to a benzoic acid of the form:

wherein R² is hydrogen, and R³ and R⁴ are independently hydrogen, C₁-C₆alkyl, C₂-C₆ alkenyl or C₂-C₆ alkynyl

by the following methodology:

A mixture of the 5-substituted 2-aminothiophenol (4.0 mmol), the benzoicacid (4.0 mmol), and polyphosphoric acid (PPA) (10 g) is heated to 220°C. for 4 hours. The reaction mixture is cooled to room temperature andpoured into 10% potassium carbonate solution (˜400 mL). The precipitateis collected by filtration under reduced pressure to give the desiredproduct, which can be purified by flash chromatography orrecrystallization.

The R² hydrogen can be substituted with either a non-radioactive halo ora radioactive halo by the following reaction:

To a solution of 6-substituted 2-(4′-aminophenyl)-benzothiazole (1 mg)in 250 μL acetic acid in a sealed vial is added 40 μL of chloramine-Tsolution (28 mg dissolved in 500 μL acetic acid) followed by 27 μL (ca.5 mCi) of sodium [¹²⁵I]iodide (specific activity 2,175 Ci/mmol). Thereaction mixture is stirred at room temperature for 2.5 hours andquenched with saturated sodium hydrogensulfite solution. After dilutionwith 20 ml of water, the reaction mixture is loaded onto C8 Plus SepPakand eluted with 2 ml methanol. Depending on the nature of thesubstituent on the 6-position, protecting groups may need to beemployed. For example, the 6-hydroxy group is protected as themethanesulfonyl (mesyloxy) derivative. For deprotection of themethanesulfonyl group, 0.5 ml of 1 M NaOH is added to the elutedsolution of radioiodinated intermediate. The mixture is heated at 50° C.for 2 hours. After being quenched by 500 μL of 1 M acetic acid, thereaction mixture is diluted with 40 mL of water and loaded onto a C8Plus SepPak. The radioiodinated product, having a radioactivity of ca. 3mCi, is eluted off the SepPak with 2 mL of methanol. The solution iscondensed by a nitrogen stream to 300 μL and the crude product ispurified by HPLC on a Phenomenex ODS column (MeCN/TEA buffer, 35:65, pH7.5, flow rate 0.5 mL/minute up to 4 minutes, 1.0 mL/minute at 4-6minutes, and 2.0 mL/minute after 6 minutes, retention time 23.6). Thecollected fractions are loaded onto a C8 Plus SepPak. Elution with 1 mLof ethanol gave ca. 1 mCi of the final radioiodinated product.

When either or both R³ and R⁴ are hydrogen, then R³ and R⁴ can beconverted to C₁-C₆ alkyl, C₂-C₆ alkenyl or C₂-C₆ alkynyl by reactionwith an alkyl, alkenyl or alkynyl halide under the following conditions:

For dialkylation: To a solution of 6-substituted2-(4′-aminophenyl)-benzothiazole (0.59 mmol) in DMSO (anhydrous, 2 ml)are added alkyl, alkenyl, or alkynyl halide (2.09 mmol), and K₂CO₃ (500mg, 3.75 mmol). The reaction mixture is heated at 140° C. for 16 hours.Upon cooling to room temperature, the reaction mixture is poured intowater and extracted with ethyl acetate (3×10 mL). The organic layers arecombined and the solvent is evaporated. The residue is purified by flashcolumn to give the desired 6-substituteddimethylaminophenyl)-benzothiazole.

For monoalkylation: To a solution of 6-substituted2-(4′-aminophenyl)-benzothiazole (0.013 mmol) in DMSO (anhydrous, 0.5ml) is added alkyl, alkenyl, or alkynyl halide (0.027 mmol) andanhydrous K₂CO₃ (100 mg, 0.75 mmol). The reaction mixture is heated at100° C. for 16 hours. Upon cooling to room temperature, the reactionmixture is directly purified by normal phase preparative TLC to give thedesired 6-substituted-2-(4′-methylaminophenyl)-benzothiazolederivatives.

When R² is hydrogen or a non-radioactive halo, R⁴ is C₁-C₆ alkyl, C₂-C₆alkenyl or C₂-C₆ alkynyl, wherein the alkyl, alkenyl or alkynylcomprises a radioactive carbon or is substituted with a radioactivehalo, the compound can be synthesized by one of the following sequences:

For Radioactive Carbon Incorporation:

Approximately 1 Ci of [¹¹C]carbon dioxide is produced using aCTI/Siemens RDS 112 negative ion cyclotron by irradiation of a nitrogengas (¹⁴N₂) target containing 1% oxygen gas with a 40 μA beam current of11 MeV protons for 60 minutes. [¹¹C]Carbon dioxide is converted to[¹¹C]methyl iodide by first reacting it with a saturated solution oflithium aluminum hydride in THF followed by the addition of hydriodicacid at reflux temperature to generate [¹¹C]methyl iodide. The[¹¹C]methyl iodide is carried in a stream of nitrogen gas to a reactionvial containing the precursor for radiolabeling. The precursor,6-substituted 2-(4′-aminophenyl)-benzothiazole (˜17 mmoles), isdissolved in 400 μL of DMSO. Dry KOH (10 mg) is added, and the 3 mLV-vial is vortexed for 5 minutes. No-carrier-added [¹¹C]methyl iodide isbubbled through the solution at 30 mL/minute at room temperature. Thereaction is heated for 5 minutes at 95° C. using an oil bath. Thereaction product is purified by semi-preparative HPLC using a ProdigyODS-Prep column eluted with 60% acetonitrile/40% triethylammoniumphosphate buffer pH 7.2 (flow at 5 mL/minute for 0-7 minutes thenincreased to 15 mL/minute for 7-30 minutes). The fraction containing[N-methyl-¹¹C]6-substituted 2-(4′-methylaminophenyl)-benzothiazole (atabout 15 min) is collected and diluted with 50 mL of water and elutedthrough a Waters C18 SepPak Plus cartridge. The C18 SepPak is washedwith 10 mL of water, and the product is eluted with 1 mL of ethanol(absolute) into a sterile vial followed by 14 mL of saline.Radiochemical and chemical purities are >95% as determined by analyticalHPLC (k′=4.4 using the Prodigy ODS(3) analytical column eluted with65/35 acetonitrile/triethylammonium phosphate buffer pH 7.2). Theradiochemical yield averages 17% at EOS based on [¹¹C]methyl iodide, andthe specific activity averages about 160 GBq/μmol (4.3 Ci/μmol) at endof synthesis.

For radioactive halogen incorporation:

A mixture of 6-substituted 2-(4′-aminophenyl)-benzathiazole (protectinggroups may be necessary depending on the nature of the 6-substituent asnoted above) (0.22 mmol), NaH (4.2 mmol) and2-(-3-bromopropoxy)tetrahydro-2-H-pyran (0.22 mmol) in THF (8 mL) isheated to reflux for 23 hours. The solvent is removed by distillationand the residue is dissolved in to ethyl acetate and water, the organiclayer is separated and the aqueous layer is extracted with ethyl acetate(10 mL×6). The organic layer is combined and dried over MgSO₄ andevaporated to dryness. The residue is added AcOH/THF/H₂O solution (5 mL,4/2/1) and heated to 100° C. for 4 hours. The solvent is removed byevaporation and the residue is dissolved in ethyl acetate (˜10 mL)washed by NaHCO₃ solution, dried over MgSO₄ and evaporated to dryness togive a residue which is purified with preparative TLC(hexane:ethylacetate=60:40) to give the desired 6-substituted2-(4′-(3″-hydroxypropylamino)-phenyl)-benzothiazole (45%).

To a solution of 6-substituted2-(4′-(3″-hydroxypropylamino)-phenyl)-benzathiazole (0.052 mmol) andEt₃N (0.5 ml) dissolved in acetone (5 mL) is added (Boc)₂O (50 mg, 0.22mmol). The reaction mixture is stirred at room temperature for 6 hoursfollowed by addition of tosyl chloride (20 mg, 0.11 mmol). The reactionmixture is stirred at room temperature for another 24 hours. The solventis removed and the residue is dissolved into ethyl acetate (10 mL),washed with NaCO₃ solution, dried over MgSO₄, evaporated, and purifiedwith flash column (Hexane/ethyl acetate=4/1) to give the desired6-substituted2-(4′-(3″-toluenesulfonoxypropylamino)-phenyl)-benzothiazole (13%). This6-substituted2-(4′-(3″-toluenesulfonoxypropylamino)-phenyl)-benzothiazole is thenradiofluorinated by standard methods as follows:

A cyclotron target containing 0.35 mL of 95% [O-18]-enriched water isirradiated with 11 MeV protons at 20 μA of beam current for 60 minutes,and the contents are transferred to a 5 mL reaction vial containingKryptofix 222 (22.3 mg) and K₂CO₃ (7.9 mg) in acetonitrile (57 μL). Thesolution is evaporated to dryness three times at 110° C. under a streamof argon following the addition of 1 mL aliquots of acetonitrile. To thedried [F-18]fluoride is added 3 mg of 6-substituted2-(4′-(3″-toluenesulfonoxypropylamino)-phenyl)-benzothiazole in 1 mLDMSO, and the reaction vial is sealed and heated to 85° C. for 30minutes. To the reaction vial, 0.5 mL of MeOH/HCl (concentrated) (2/1v/v) is added, and the vial is heated at 120° C. for 10 minutes. Afterheating, 0.3 mL of 2 M sodium acetate buffer is added to the reactionsolution followed by purification by semi-prep HPLC using a PhenomenexProdigy ODS-prep C18 column (10 μm 250×10 mm) eluted with 40%acetonitrile/60% 60 mM triethylamine-phosphate buffer (v/v) pH 7.2 at aflow rate of 5 mL/minute for 15 minutes, then the flow is increased to 8mL/minute for the remainder of the separation. The product,[F-18]6-substituted 2-(4′-(3″-fluoropropylamino)-phenyl)-benzothiazole,is eluted at ˜20 minutes in a volume of about 16 mL. The fractioncontaining [F-18]6-substituted2-(4′-(3″-fluoropropylamino)-phenyl)-benzothiazole is diluted with 50 mLof water and eluted through a Waters C18 SepPak Plus cartridge. TheSepPak cartridge is then washed with 10 mL of water, and the product iseluted using 1 mL of ethanol (absol.) into a sterile vial. The solutionis diluted with 10 mL of sterile normal saline for intravenous injectioninto animals. The [F-18]6-substituted2-(4′-(3″-fluoropropylamino)-phenyl)-benzothiazole product is obtainedin 2-12% radiochemical yield at the end of the 120 minute radiosynthesis(not decay corrected) with an average specific activity of 1500 Ci/mmol.

Example 1 [N-Methyl-¹¹C]2-(4%Dimethylaminophenyl)-6-methoxy-benzothiazole was synthesized accordingto Scheme I

Approximately 1 Ci of [¹¹C]carbon dioxide was produced using aCTI/Siemens RDS 112 negative ion cyclotron by irradiation of a nitrogengas (¹⁴N₂) target containing 1% oxygen gas with a 40 μA beam current of11 MeV protons for 60 minutes. [¹¹C]Carbon dioxide is converted to[¹¹C]methyl iodide by first reacting it with a saturated solution oflithium aluminum hydride in THF followed by the addition of hydriodicacid at reflux temperature to generate [¹¹C]methyl iodide. The[¹¹C]methyl iodide is carried in stream of nitrogen gas to a reactionvial containing the precursor for radiolabeling. The precursor,6-CH₃O-BTA-1 (1.0 mg, 3.7 μmoles), was dissolved in 400 μL of DMSO. DryKOH (10 mg) was added, and the 3 mL V-vial was vortexed for 5 minutes.No-carrier-added [¹¹C]methyl iodide was bubbled through the solution at30 mL/minute at room temperature. The reaction was heated for 5 minutesat 95° C. using an oil bath. The reaction product was purified bysemi-preparative HPLC using a Prodigy ODS-Prep column eluted with 60%acetonitrile/40% triethylammonium phosphate buffer pH 7.2 (flow at 5mL/minute for 0-7 minutes then increased to 15 mL/minute for 7-30minutes). The fraction containing[N-Methyl-¹¹C]2-(4′-Dimethylaminophenyl)-6-methoxy-benzothiazole (atabout 15 minutes) was collected and diluted with 50 mL of water andeluted through a Waters C18 SepPak Plus cartridge. The C18 SepPak waswashed with 10 mL of water, and the product was eluted with 1 mL ofethanol (absolute) into a sterile vial followed by 14 mL of saline.Radiochemical and chemical purities were >95% as determined byanalytical HPLC (k′=4.4 using the Prodigy ODS(3) analytical columneluted with 65/35 acetonitrile/triethylammonium phosphate buffer pH7.2). The radiochemical yield averaged 17% at EOS based on [¹¹C]methyliodide, and the specific activity averaged about 160 GBq/μmol (4.3Ci/μmol) at end of synthesis.

Example 2 2-(3′-¹²⁵I-iodo-4′-amino-phenyl)-benzothiazol-6-ol wassynthesized according to Scheme II

To a solution of 2-(4′-aminophenyl)-6-methanesulfonoxy-benzothiazole (1mg) in 250 μL acetic acid in a sealed vial was added 40 μL of chloramineT solution (28 mg dissolved in 500 μL acetic acid) followed by 27 μL(ca. 5 mCi) of sodium [¹²⁵I]iodide (specific activity 2,175 Ci/mmol).The reaction mixture was stirred at room temperature for 2.5 hours andquenched with saturated sodium hydrogensulfite solution. After dilutionwith 20 ml of water, the reaction mixture was loaded onto C8 Plus SepPakand eluted with 2 ml methanol. For deprotection of the methanesulfonylgroup, 0.5 ml of 1 M NaOH was added to the eluted solution ofradioiodinated intermediate. The mixture was heated at 50° C. for 2hours. After being quenched by 500 μL of 1 M acetic acid, the reactionmixture was diluted with 40 mL of water and loaded onto a C8 PlusSepPak. The radioiodinated product, having a radioactivity of ca. 3 mCi,was eluted off the SepPak with 2 mL of methanol. The solution wascondensed by a nitrogen stream to 300 μL and the crude product waspurified by HPLC on a Phenomenex ODS column (MeCN/TEA buffer, 35:65, pH7.5, flow rate 0.5 mL/minute up to 4 minutes, 1.0 mL/minute at 4-6minutes, and 2.0 mL/minute after 6 minutes, retention time 23.6). Thecollected fractions were loaded onto a C8 Plus SepPak. Elution with 1 mLof ethanol gave ca. 1 mCi of the final radioiodinated product.

Preparation of the ¹²³I radiolabeled derivatives, proceeds similarly tothe synthesis outlined above. For example, replacing sodium [¹²⁵I]iodidewith sodium [¹²³I]iodide in the synthetic method would provide the ¹²³Iradiolabeled compound. Such substitution of one radiohalo atom foranother is well known in the art, see for example, Mathis C A, Taylor SE, Biegon A, Enas J D. [¹²⁵I]5-Iodo-6-nitroquipazine: a potent andselective ligand for the 5-hydroxytryptamine uptake complex I. In vitrostudies. Brain Research 1993; 619:229-235; Jagust W, Eberling J L,Roberts J A, Brennan K M, Hanrahan S M, Van Brocklin H, Biegon A, MathisC A. In vivo imaging of the 5-hydroxytryptamine reuptake site in primatebrain using SPECT and [¹²³I]5-iodo-6-nitroquipazine. European Journal ofPharmacology 1993; 242:189-193; Jagust W J, Eberling J L, Biegon A,Taylor S E, VanBrocklin H, Jordan S, Hanrahan S M, Roberts J A, BrennanK M, Mathis C A. [Iodine-123]5-Iodo-6-Nitroquipazine: SPECT Radiotracerto Image the Serotonin Transporter. Journal of Nuclear Medicine 1996;37:1207-1214.)

Example 3 2-(3-¹⁸F-Fluoro-4-methylamino-phenyl)-benzothiazol-6-ol wassynthesized according to Scheme III

A cyclotron target containing 0.35 mL of 95% [O-18]-enriched water wasirradiated with 11 MeV protons at 20 μA of beam current for 60 minutes,and the contents were transferred to a 5 mL reaction vial containing 2mg Cs₂CO₃ in acetonitrile (57 μL). The solution was evaporated todryness at 110° C. under a stream of argon three times using 1 mLaliquots of acetonitrile. To the dried [F-18]fluoride was added 6 mg of6-MOMO-BT-3′-Cl-4′-NO₂ in 1 mL DMSO, and the reaction vial was sealedand heated to 120° C. for 20 minutes (radiochemical incorporation forthis first radiosynthesis step was about 20% of solubilized[F-18]fluoride). To the crude reaction mixture was added 8 mL of waterand 6 mL of diethyl ether, the mixture was shaken and allowed toseparate. The ether phase was removed and evaporated to dryness under astream of argon at 120° C. To the dried sample, 0.5 mL of absolute EtOHwas added along with 3 mg copper (II) acetate and 8 mg of NaBH₄. Thereduction reaction was allowed to proceed for 10 minutes at roomtemperature (the crude yield for the reduction step was about 40%). Tothe reaction mixture was added 8 mL of water and 6 mL of diethyl ether,the mixture was shaken and the ether phase separated. The diethyl etherphase was dried under a stream of argon at 120° C. To the reaction vial,700 uL of DMSO was added containing 30 micromoles of CH₃I and 20 mg ofdry KOH. The reaction vial was heated at 120° C. for 10 minutes. Asolution of 700 uL of 2:1 MeOH/HCl (concentrated) was added and heatedfor 15 minutes at 120° C. After heating, 1 mL of 2 M sodium acetatebuffer was added to the reaction solution followed by purification bysemi-prep HPLC using a Phenomenex Prodigy ODS-prep C18 column (10 μm250×10 mm) eluted with 35% acetonitrile/65% 60 mMtriethylamine-phosphate buffer (v/v) pH 7.2 at a flow rate of 5mL/minute for 2 minutes, then the flow was increased to 15 mL/minute forthe remainder of the separation. The product,2-(3-¹⁸F-fluoro-4-methylamino-phenyl)-benzothiazol-6-ol, eluted at ˜15minutes in a volume of about 16 mL. The fraction containing2-(3-¹⁸F-fluoro-4-methylamino-phenyl)-benzothiazol-6-ol was diluted with50 mL of water and eluted through a Waters C18 SepPak Plus cartridge.The SepPak cartridge was then washed with 10 mL of water, and theproduct was eluted using 1 mL of ethanol (absol.) into a sterile vial.The solution was diluted with 10 mL of sterile normal saline forintravenous injection into animals. The2-(3-¹⁸F-fluoro-4-methylamino-phenyl)-benzothiazol-6-ol product wasobtained in 0.5% (n=4) radiochemical yield at the end of the 120 minuteradiosynthesis (not decay corrected) with an average specific activityof 1000 Ci/mmol. The radiochemical and chemical purities of2-(3-¹⁸F-fluoro-4-methylamino-phenyl)-benzothiazol-6-ol were assessed byradio-HPLC with UV detection at 350 nm using a Phenomenex Prodigy ODS(3)C18 column (5 μm, 250×4.6 mm) eluted with 40% acetonitrile/60% 60 mMtriethylamine-phosphate buffer (v/v) pH 7.2.2-(3-¹⁸F-Fluoro-4-methylamino-phenyl)-benzothiazol-6-ol had a retentiontime of ˜11 minutes at a flow rate of 2 mL/min (k′=5.5). Theradiochemical purity was >99%, and the chemical purity was >90%. Theradiochemical identity of2-(3-¹⁸F-Fluoro-4-methylamino-phenyl)-benzothiazol-6-ol was confirmed byreverse phase radio-HPLC utilizing a quality control sample of the finalradiochemical product co-injected with a authentic (cold) standard.

Example 4 2-[4-(3-¹⁸F-Fluoro-propylamino)-phenyl]-benzothiazol-6-ol wassynthesized according to Scheme IV

A cyclotron target containing 0.35 mL of 95% [O-18]-enriched water wasirradiated with 11 MeV protons at 20 μA of beam current for 60 minutes,and the contents were transferred to a 5 mL reaction vial containingKryptofix 222 (22.3 mg) and K₂CO₃ (7.9 mg) in acetonitrile (57 μL). Thesolution was evaporated to dryness three times at 110° C. under a streamof argon following the addition of 1 mL aliquots of acetonitrile. To thedried [F-18]fluoride was added 3 mg of 6-MOMO-BTA-N-Pr-Ots in 1 mL DMSO,and the reaction vial was sealed and heated to 85° C. for 30 minutes. Tothe reaction vial, 0.5 mL of MeOH/HCl (concentrated) (2/1 v/v) wasadded, and the vial was heated at 120° C. for 10 minutes. After heating,0.3 mL of 2 M sodium acetate buffer was added to the reaction solutionfollowed by purification by semi-prep HPLC using a Phenomenex ProdigyODS-prep C18 column (10 μm 250×10 mm) eluted with 40% acetonitrile/60%60 mM triethylamine-phosphate buffer (v/v) pH 7.2 at a flow rate of 5mL/minute for 15 minutes, then the flow was increased to 8 mL/minute forthe remainder of the separation. The product, [F-18]6-HO-BTA-N-PrF,eluted at ˜20 minutes in a volume of about 16 mL. The fractioncontaining [F-18]6-HO-BTA-N-PrF was diluted with 50 mL of water andeluted through a Waters C18 SepPak Plus cartridge. The SepPak cartridgewas then washed with 10 mL of water, and the product was eluted using 1mL of ethanol (absol.) into a sterile vial. The solution was dilutedwith 10 mL of sterile normal saline for intravenous injection intoanimals. The [F-18]6-HO-BTA-N-PrF product was obtained in 8±4% (n=8)radiochemical yield at the end of the 120 minute radiosynthesis (notdecay corrected) with an average specific activity of 1500 Ci/mmol. Theradiochemical and chemical purities of [F-18]6-HO-BTA-N-PrF wereassessed by radio-HPLC with UV detection at 350 nm using a PhenomenexProdigy ODS(3) C18 column (5 μm, 250×4.6 mm) eluted with 40%acetonitrile/60% 60 mM triethylamine-phosphate buffer (v/v) pH 7.2.[F-18]6-HO-BTA-N-PrF had a retention time of ˜12 minutes at a flow rateof 2 mL/minute (k′=6.1). The radiochemical purity was >99%, and thechemical purity was >90%. The radiochemical identity of[F-18]6-HO-BTA-N-PrF was confirmed by reverse phase radio-HPLC utilizinga quality control sample of the final radiochemical product co-injectedwith a authentic (cold) standard.

Example 5 Synthesis of 2-(3′-iodo-4′-aminophenyl)-6-hydroxybenzothiazole

Preparation of 4-Methoxy-4′-nitrobenzanilide

p-Anisidine (1.0 g, 8.1 mmol) was dissolved in anhydrous pyridine (15ml), 4-nitrobenzoyl chloride (1.5 g, 8.1 mmol) was added. The reactionmixture was allowed to stand at room temperature for 16 hrs. Thereaction mixture was poured into water and the precipitate was collectedwith filtrate under vacuum pressure and washed with 5% sodiumbicarbonate (2×10 ml). The product was used in the next step withoutfurther purification. ¹HNMR (300 MHz, DMSO-d₆) δ: 10.46(s, 1H, NH),8.37(d, J=5.5 Hz, 2H, H-3′,5′), 8.17(d, J=6.3 Hz, 2H, H-2′,6′), 7.48(d,J=6.6 Hz, 2H), 6.97(d, J=6.5 Hz, 2H), 3.75(s, 3H, MeO).

Preparation of 4-Methoxy-4′-nitrothiobenzanilide

A mixture of 4-methoxy-4′-nitrothiobenzaniline (1.0 g, 3.7 mmol) andLawesson's reagent (0.89 g, 2.2 mmol, 0.6 equiv.) in chlorobenzene (15mL) was heated to reflux for 4 hrs. The solvent was evaporated and theresidue was purified with flush column (hexane:ethyl acetate=4:1) togive 820 mg (77.4%) of the product as orange color solid. ¹HNMR (300MHz, DMSO-d₆) δ: 8.29(d, 2H, H-3′,5′), 8.00(d, J=8.5 Hz, 2H, H-2′,6′),7.76(d, 2H), 7.03(d, J=8.4 Hz, 2H), 3.808.37(d, J=5.5 Hz, 2H, H-3′,5′),8.17(d, J=6.3 Hz, 2H, H-2′,6′), 7.48(d, J=6.6 Hz, 2H), 6.97(d, J=6.5 Hz,2H), 3.75(s, 3H, MeO). (s, 3H, MeO).

Preparation of 6-Methoxy-2-(4-nitrophenyl)benzothiazole

4-Methoxy-4′-nitrothiobenzanilides (0.5 g, 1.74 mmol) was wetted with alittle ethanol (˜0.5 mL), and 30% aqueous sodium hydroxide solution (556mg 13.9 mmol. 8 equiv.) was added. The mixture was diluted with water toprovide a final solution/suspension of 10% aqueous sodium hydroxide.Aliquots of this mixture were added at 1 min intervals to a stirredsolution of potassium ferricyanide (2.29 g, 6.9 mmol, 4 equiv.) in water(5 mL) at 80-90° C. The reaction mixture was heated for a further 0.5 hand then allowed to cool. The participate was collected by filtrationunder vacuum pressure and washed with water, purified with flush column(hexane:ethyl acetate=4:1) to give 130 mg (26%) of the product. ¹HNMR(300 MHz, Acetone-d₆) δ: 8.45(m, 4H), 8.07(d, J=8.5 Hz, 1H, H-4),7.69(s, 1H, H-7), 7.22(d, J=9.0 Hz, 1H, H-5), 3.90(s, 3H, MOD)

Preparation of 6-Methoxy-2-(4-aminophenyl)benzothiazole

A mixture of the 6-methoxy-2-(4-nitropheyl)benzothiazoles (22 mg, 0.077mmol) and tin(II) chloride (132 mg, 0.45 mmol) in boiling ethanol wasstirred under nitrogen for 4 hrs. Ethanol was evaporated and the residuewas dissolved in ethyl acetate (10 mL), washed with 1 N sodium hydroxide(2 mL) and water (5 mL), and dried over MgSO₄. Evaporation of thesolvent gave 19 mg (97%) of the product as yellow solid.

Preparation of 2-(3′-iodo-4′-aminophenyl)-6-methoxybenzothiazole

To a solution of 2-(4′-aminophenyl)-6-methoxy benzothiazole (22 mg, 0.09mmol) in glacial acetic acid (2.0 mL) was injected 1 M iodochloridesolution in CH₂Cl₂ (0.10 mL, 0.10 mmol, 1.2 eq.) under N₂ atmosphere.The reaction mixture was stirred at room temperature for 16 hr. Theglacial acetic acid was removed under reduced pressure and the residuewas dissolved in CH₂Cl₂. After neutralizing the solution with NaHCO₃,the aqueous layer was separated and extracted with CH₂Cl₂. The organiclayers were combined and dried over MgSO₄. Following the evaporation ofthe solvent, the residue was purified by preparative TLC(Hexanes:ethylacetate=6:1) to give 2-(4′-amino-3′-iodophenyl)-6-methoxy benzothiazole(25 mg, 76%) as brown solid. ¹HNMR (300 MHz, CDCl₃) δ (ppm): 8.35 (d,J=2.0 Hz, 1H), 7.87 (dd, J₁=2.0 Hz, J₂=9.0 Hz, 1H), 7.31 (d, J=2.2 Hz,1H), 7.04 (dd, J₁=2.2 Hz, J₂=9.0 Hz, 1H), 6.76 (d, J=9.0 Hz, 1H), 3.87(s, 3H).

Preparation of 2-(3′-Iodo-4′-aminophenyl)-6-hydroxybenzothiazole

To a solution of 2-(4′-Amino-3′-iodophenyl)-6-methoxy benzothiazole (5)(8.0 mg, 0.02 mmol) in CH₂Cl₂ (2.0 mL) was injected 1 M BBr₃ solution inCH₂Cl₂ (0.20 ml, 0.20 mmol) under N₂ atmosphere. The reaction mixturewas stirred at room temperature for 18 hrs. After the reaction wasquenched with water, the mixture was neutralized with NaHCO₃. Theaqueous layer was extracted with ethyl acetate (3×3 mL). The organiclayers were combined and dried over MgSO₄. The solvent was thenevaporated under reduced pressure and the residue was purified bypreparative TLC (Hexanes:ethyl acetate=7:3) to give2-(3′-iodo-4′-aminophenyl)-6-hydroxybenzothiazole (4.5 mg, 58%) as abrown solid. ¹HNMR (300 MHz, acetone-d₆) δ (ppm): 8.69 (s, 1H), 8.34 (d,J=2.0 Hz, 1H), 7.77 (dd, J₁=2.0 Hz, J₂=8.4 Hz, 1H), 7.76 (d, J=8.8 Hz,1H), 7.40 (d, J=2.4 Hz, 1H), 7.02 (dd, J₁=2.5 Hz, J₂=8.8 Hz, 1H), 6.94(d, J=8.5 Hz, 1H), 5.47 (br., 2H). HRMS m/z 367.9483 calcd forC₁₃H₉N₂OSI 367.9480).

Example 6 Synthesis of2-(3′-iodo-4′-methylaminophenyl)-6-hydroxybenzothiazole

Preparation of 6-Methoxy-2-(4-methylaminophenyl)benzothiazole

A mixture of 4-methylaminobenzoic acid (11.5 g, 76.2 mmol) and5-methoxy-2-aminothiophenol (12.5, g, 80 mmol) was heated in PPA (˜30 g)to 170° C. under N₂ atmosphere for 1.5 hr. The reaction mixture was thencooled to room temperature and poured into 10% K₂CO₃ solution. Theprecipitate was filtered under reduced pressure. The crude product wasre-crystallized twice from acetone/water and THF/water followed by thetreatment with active with carbon to give 4.6 g (21%) of6-Methoxy-2-(4-methylaminophenyl)benzothiazole as a yellow solid. ¹HNMR(300 MHz, acetone-d₆) δ: 7.84(d, J=8.7 Hz, 2H, H-2′6′), 7.78(dd, J₁=8.8Hz, J₂=1.3 Hz, 1H, H-4), 7.52(d, J=2.4 Hz, 1H, H-7), 7.05(dd, J₁=8.8 Hz,J₂=2.4 Hz, H-5), 6.70(d, J=7.6 Hz, 2H, H-3′5′), 5.62(s, 1H, NH), 3.88(s,3H, OCH₃), 2.85(d, J=6.2 Hz, 3H, NCH₃)

Preparation of 2-(3′-Iodo-4′-methylaminophenyl)-6-methoxy benzothiazole

To a solution of 2-(4′-Methylaminophenyl)-6-methoxy benzothiazole (20mg, 0.074 mmol) dissolved in glacial acetic acid (2 mL) was added Icl(90 μL, 0.15 mmol, 1.2 eq, 1M in CH₂Cl₂) under N₂. The reaction wasallowed to stir at room temperature for 18 hr. The glacial acetic acidwas then removed under reduced pressure. The residue was dissolved inCH₂Cl₂ and neutralized with NaHCO₃. The aqueous layer was extracted withCH₂Cl₂ and the organic layers were combined, dried over MgSO₄ andevaporated. The residue was purified with preparative TLC(Hexane:EA=2:1) to give 2-(4′-methylamino-3′-iodophenyl)-6-methoxybenzothiazole (8 mg, 27%) as brown solid. ¹HNMR (300 MHz, CDCl₃) δ(ppm):8.39(d, J=2.0 Hz, 1H), 7.88 (d, J=9.0 Hz, 1H), 7.33 (d, J=2.2 Hz, 1H),7.06 (dd, J₁=2.2 Hz, J₂=9.0 Hz, 1H), 6.58 (d, J=9.0 Hz, 1H), 3.89 (s,3H, OCH₃).

Preparation of 2-(3′-Iodo-4′-methylamino-phenyl)-6-hydroxy benzothiazole

To a solution of 2-(4′-methylamino-3′-iodophenyl)-6-methoxybenzothiazole (12 mg, 0.03 mmol) dissolved in CH₂Cl₂ (4 mL) was addedBBr₃ (400 μl, 0.4 mmol, 1M in CH₂Cl₂) under N₂. The reaction was allowedto stir at room temperature for 18 hr. Water was then added to quenchthe reaction and the solution was neutralized with NaHCO₃, extractedwith ethyl acetate (3×5 mL). The organic layers were combined, driedover MgSO₄ and evaporated. The residue was purified with preparative TLC(Hexane:EA=7:3) to give 2-(4′-methylamino-3′-iodophenyl)-6-hydroxybenzothiazole (5 mg, 43%) as brown solid. ¹HNMR (300 MHz, CDCl₃) δ(ppm):8.37 (d, H=2.0 Hz, 1H), 7.88 (dd, J₁=2.0 Hz, J₂=8.4 Hz, 1H), 7.83 (d,J=8.8 Hz, 1H), 7.28 (d, J=2.4 Hz, 1H), 6.96 (dd, J₁=2.5 Hz, J₂=8.8 Hz,1H), 6.58 (d, J=8.5 Hz, 1H), 2.96 (s, 3H, CH₃).

Example 7 Radiosynthesis of [¹²⁵I]6-OH-BTA-0-3′-I

Preparation of 2-(4′-Nitrophenyl)-6-hydroxybenzothiazole

To a suspension of 2-(4′-nitrophenyl)-6-methoxy benzothiazole (400 mg,1.5 mmol) in CH₂Cl₂ (10 mL) was added BBr₃ (1M in CH₂Cl₂, 10 mL, 10mmol). The reaction mixture was stirred at room temperature for 24 hr.The reaction was then quenched with water, and extracted with ethylacetate (3×20 mL). The organic layers were combined and washed withwater, dried over MgSO₄, and evaporated. The residue was purified byflash chromatography (silica gel, hexanes:ethyl acetate=1:1) to give theproduct as a yellow solid (210 mg, 55%). ¹HNMR (300 MHz, Acetone-d₆) δ(ppm): 9.02(s, OH), 8.41(d, J=9.1 Hz, 1H), 8.33(d, J=9.1 Hz, 1H),7.96(d, J=8.6 Hz, 1H), 7.53(d, J=2.4 Hz, 1H), 7.15(dd, J1=8.6 Hz, J2=2.4Hz, 1H).

Preparation of 2-(4′-Nitrophenyl)-6-methylsulfoxy benzothiazole

To a solution of 2-(4′-nitrophenyl)-6-hydroxy benzothiazole (50 mg, 0.18mmol) dissolved in acetone (7 mL, anhydrous) was added K₂CO₃ (100 mg,0.72 mmol, powdered) and MsCl (200 ul). After stirring for 2 hrs, thereaction mixture was filtered. The filtrate was concentrated and theresidue was purified by flash column (silica gel, hexane:ethylacetate=4:1) to give 2-(4-nitrophenyl)-6-methylsulfoxy benzothiazole (44mg, 68%) as pale yellow solid. ¹HNMR (300 MHz, acetone-d₆) δ (ppm):8.50-8.40(m, 4H), 8.29(d, J=2.3 Hz, 1H), 8.23(d, J=8.9 Hz, 1H), 7.61(dd,J₁=2.3 Hz, J₂=8.9 Hz, 1H).

Preparation of 2-(4′-Aminophenyl)-6-methylsulfoxy benzothiazole

To a solution of 2-(4′-nitrophenyl)-6-methylsulfoxy benzothiazole (35mg, 0.10 mmol) dissolved in ethanol (10 mL) was added SnCl₂.2H₂O (50mg). The reaction mixture was heated to reflux for 1.5 hr. The solventwas then removed under reduced pressure. The residue was dissolved inethyl acetate (10 mL), washed with 1N NaOH, water, dried over MgSO₄.Evaporation of the solvent afforded 2-(4′-aminophenyl)-6-methylsulfoxybenzothiazole (21 mg, 65%) as pale brown solid. ¹HNMR (300 MHz, CDCl₃) δ(ppm): 8.02(d, J=6.2 Hz, 1H), 7.92(d, J=8.7 Hz, 2H), 7.84(d, J=2.4 Hz,1H), 7.38(dd, J₁=2.4 Hz, J₂=6.2 Hz, 1H), 6.78(d, J=8.7 Hz, 2H), 2.21(s,3H, CH₃).

Example 8 Radiosynthesis of [¹²⁵I]6-OH-BTA-1-3′-I

To a solution of 2-(4′-methylaminophenyl)-6-hydroxy benzothiazole (300mg, 1.17 mmol) dissolved in CH₂Cl₂ (20 mL) was added Et₃N (2 mL) andtrifluoroacetic acid (1.5 mL). The reaction mixture was stirred at roomtemperature for 3 h. The solvent was removed under reduced pressure andthe residue was dissolved in ethyl acetate (30 mL), washed with NaHCO₃solution. Brine, water, and dried over MgSO₄. After evaporation of thesolvent, the residue was dissolved in acetone (20 ml, pre-dried overK₂CO₃), K₂CO₃ (1.0 g, powered) was added followed by MsCl (400 mg, 3.49mmol). The reaction mixture was stirred at room temperature andmonitored with TLC omog starting material disappeared. The residue wasthen filtrated. The filtrate was evaporated under reduced pressure. Theresidue was dissolved in ethyl acetate (30 mL), washed with NaHCO₃solution. Brine, water, and dried over MgSO₄. After evaporation of thesolvent, the residue was dissolved in EtOH and NaBH₄ was added. Thereaction mixture was stirred at room temperature for 2 h. The solventwas evaporated and the residue was dissolved in water, extracted withethyl acetate (20 ml×3), the extracts were combined and dried overMgSO₄. After evaporation of the solvent, the residue was purified withflash column (hexanes/ethyl acetate=8:1) to give the product (184 mg,47.0%) as brown solid. ¹HNMR (300 MHz, CDCl₃) δ (ppm): 7.94(d, J=8.8 Hz,1H), 7.87(d, J=8.7 Hz, 2H), 7.77(d, J=2.3 Hz, 1H), 7.30(dd, J₁=8.8 Hz,J₂=2.3 Hz, 1H), 6.63(d, J=8.7 Hz, 2H), 3.16(s, CH₃), 2.89(s, NCH₃).

General Procedures for Radiolabelling:

To a solution of 2-(4′-aminophenyl)-6-methanesulfonoxy benzothiazole or2-(4′-methylaminophenyl)-6-methylsulfoxy benzothiazole (1 mg) in 250 μLacetic acid in a sealed vial was added 40 mL of chloramines T solution(28 mg dissolved in 500 μL acetic acid) followed by 27 mL (ca. 5 mCi) ofsodium [¹²⁵I]iodide (specific activity 2,175 Ci/mmol). The reactionmixture was stirred at r.t. for 2.5 hrs and quenched with saturatedsodium hydrogensulfite solution. After dilution with 20 ml of water, thereaction mixture was loaded onto C8 Plus SepPak and eluted with 2 mlmethanol. For deprotection of the methanesulfonyl group, 0.5 ml of 1 MNaOH was added to the eluted solution of radioiodinated intermediate.The mixture was heated at 50° C. for 2 hours. After being quenched by500 μL of 1 M acetic acid, the reaction mixture was diluted with 40 mLof water and loaded onto a C8 Plus SepPak. The radioiodinated product,having a radioactivity of ca. 3 mCi, was eluted off the SepPak with 2 mLof methanol. The solution was condensed by a nitrogen stream to 300 mLand the crude product was purified by HPLC on a Phenomenex ODS column(MeCN/TEA buffer, 35:65, pH 7.5, flow rate 0.5 mL/min up to 4 min, 1.0mL/min at 4-6 min, and 2.0 mL/min after 6 min, retention time 23.6). Thecollected fractions were loaded onto a C8 Plus SepPak. Elution with 1 mLof ethanol gave ca. 1 mCi of the final radioiodinated product.

Example 9 Treatment with AN-1792 Vaccine Decreases the Binding of theAmyloid Tracer, PIB, to Brain Homogenates

The benzothiazole amyloid-imaging PET tracer{N-methyl-¹¹C}2-[4′-(methylamino)phenyl]6-hydroxybenzothiazole(“[¹¹C]PIB”) shows a clear difference in retention between AD patientsand control subjects. This [¹¹C]PIB retention follows the knowntopography of amyloid deposition in AD brain (Klunk et al. 2004, Ann.Neurol., 55(3):306-19). To determine whether the present benzothiazoleamyloid imaging probes are sensitive to changes in brain amyloiddeposition caused by an anti-amyloid therapy in general, studies wereperformed for the binding of{N-methyl-³H}2-[4′-(methylamino)phenyl]6-hydroxy-benzothiazole ([³H]PIB)to homogenates of postmortem brain from two AN-1792-treated AD cases.Frozen blocks of frontal, temporal and parietal cortex and cerebellumfrom control brains (n=4), AD brains (n=5) and from two brains from theAN-1792 trial were obtained (Ferrer et al. 2004, Brain Pathology 14,11-20; Masliah et al. 2005, Neurology 64, 129-131). The blocks weresectioned (40 mm) and every second section was submitted forhistological analysis with antibodies specific for Aβ40 or Aβ42 or thefluorescent derivative of Congo red, X-34 (beta-sheet specific).Intervening sections were combined and homogenized in Tris-bufferedsaline with protease inhibitors. An aliquot was submitted for Aβ ELISAand another aliquot was assayed for [³H]PIB binding after dilution withphosphate-buffered saline (100 mg tissue, incubated with 1 nM [³H]PIB,filtered, washed and counted to determine bound [³H]PIB).

Neuropathologically, these brains were remarkable for a focal absence ofplaques in several cortical areas (FIGS. 2-4). The Masliah case (case#5180) was remarkably devoid of plaques (FIGS. 2-4) and showed basallevels of Aβ and [³H]PIB binding (FIG. 5). The Ferrer case (case #572)showed most apparent decreases in plaque deposition in the frontalcortex (FIGS. 3 and 4), which correlated with lower levels of Aβ and[³H]PIB binding (FIG. 5).

These findings support the following conclusions:

-   -   1. PIB Binding provides evidence of decreased amyloid load in        AN-1792-treated cases.    -   2. The decreases in PIB binding correlate with histological        evidence for plaque removal and with ELISA evidence for Aβ        removal.    -   3. It should be possible to detect in vivo decreases in amyloid        load that are caused by anti-amyloid therapies.        In addition, the focal nature of amyloid clearance means the        entire brain would be monitored, and PET imaging is well-suited        for this

Other embodiments of the invention will be apparent to those skilled inthe art from consideration of the specification and practice of theinvention disclosed herein. It is intended that the specification beconsidered as exemplary only, with the true scope and spirit of theinvention being indicated by the following claims.

As used herein and in the following claims, singular articles such as“a”, “an”, and “one” are intended to refer to singular or plural.

1. A method of identifying a patient as prodromal to a diseaseassociated with amyloid deposition, comprising (A) administering to thepatient in need thereof an effective amount of a radiolabeled compoundof formula (II):

or a pharmaceutically acceptable salt, of the compound, wherein: R¹ ishydrogen, —OH, —NO₂, —CN, —COOR, —OCH₂OR, C₁-C₆ alkyl, C₂-C₆ alkenyl,C₂-C₆ alkynyl, C₁-C₆ alkoxy or halo; R is C₁-C₆ alkyl; R² is hydrogen orhalo; R³ is hydrogen, C₁-C₆ alkyl, C₂-C₆ alkenyl or C₂-C₆ alkynyl; andR⁴ is hydrogen, C₁-C₆ alkyl, C₂-C₆ alkenyl or C₂-C₆ alkynyl, wherein thealkyl, alkenyl or alkynyl comprises a radioactive carbon or issubstituted with a radioactive halo when R² is hydrogen or anon-radioactive halo, provided that, when R¹ is hydrogen or —OH, R² ishydrogen and R⁴ is —¹¹CH₃, then R³ is C₂-C₆ alkyl, C₂-C₆alkenyl or C₂-C₆alkynyl, and further provided that, when R¹ is hydrogen, R² hydrogen andR⁴ is —(CH₂)₃ ¹⁸F, then R³ is C₂-C₆ alkyl, C₂-C₆ alkenyl or C₂-C₆alkynyl, (B) imaging said patient, then (C) administering to saidpatient in need thereof at least one anti-amyloid agent, (D)subsequently administering to said patient in need thereof an effectiveamount of a compound of formula (II), (E) imaging said patient, and (F)comparing levels of amyloid deposition in said patient before treatmentwith said at least one anti-amyloid agent to levels of amyloiddeposition in said patient after treatment with said at least oneanti-amyloid agent.